Nucleic acid sequence detection employing amplification probes

ABSTRACT

Methods and compositions are provided for detecting nucleic acid sequences. In particular, pairs of probes are employed, where the pair defines a substantially contiguous sequence on a target nucleic acid. Each of the pairs has a side chain which forms a stem of the two side chains which non-covalently binds and is capable of forming a cross-link upon activation, when the probes and sample nucleic acid are base paired. Cross-linking of the stems when unbound to complementary DNA is inhibited. Each of the nucleic acids is initially present as single stranded nucleic acid to allow for base pairing, so that the probes bind to homologous target nucleic acid. The assay mixture is activated to provide cross-linking, the double stranded nucleic acid melted, and the process of base pairing, activation and melting repeated, a sufficient number of cycles, to provide a detectable amount of cross-linked probes. To inhibit background cross-linking, the side chains may provide for duplex formation, where a portion of the side chain binds to a different portion of the side chain or the portion of the probe homologous to the target. Also provided are kits comprising reagents, as well as automatic devices, for carrying out the subject method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 08/577,121, filed Dec. 22,1995, patented and issued as U.S. Pat. No. 6,004,513 which is acontinuation-in-part of application Ser. No. 08/487,034, filed on Jun.7, 1995 now U.S. Pat. No. 5,767,259 which is a continuation-in-part ofapplication Ser. No. 08/364,339, filed on Dec. 27, 1994, patented andissued as U.S. Pat. No. 5,616,464 on Apr. 1, 1997.

INTRODUCTION

1. Technical Field

The field of this invention is nucleic acid sequence detection.

2. Background

The amount of information concerning the genomes of a large variety ofspecies is increasing exponentially. The availability of known sequencescreates an enormous market for the detection of particular sequencespresent as DNA or RNA, whereby one can detect the presence of genes,their transcription or mutations, such as lesions, substitutions,deletions, translocations, and the like. By knowing sequences ofinterest, one can detect a wide variety of pathogens, particularlyunicellular microorganisms and viral strains, and genetic diseasesincluding the presence of genes imparting antibiotic resistance to theunicellular microorganisms, as illustrative of only a few of theavailable possibilities. In addition, there are needs within theextensive areas of genetic counseling, forensic medicine, research, andthe like, for nucleic acid sequence detection technology.

In many instances, the target nucleic acid sequence is only a very smallproportion of total nucleic acid in the sample. Furthermore, there maybe many situations where the target nucleic acid of interest and othersequences present have substantial homology. It is therefore importantto develop methods for the detection of the target nucleic acid sequencethat are both sensitive and accurate.

Several enzymatic amplification methods have been developed, such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), NASBA,and self-sustained sequence replication (SSR). The first and mostnotable method that has received extensive use is PCR. Starting withspecific primers, nucleoside triphosphate monomers, the target strand ofDNA and a polymerase enzyme, one can greatly amplify the target DNAsequence of interest. This technology is extremely powerful and has beenapplied to a myriad of research applications, but it has a number ofdrawbacks which limit its use in a variety of areas. Generalavailability is limited by the restrictive nature of licenses by theowners of the patent rights. In addition, the method requires an enzyme.While the availability of thermally stable enzymes has greatly enhancedthe applicability of PCR, there is nevertheless the inconvenience thatdenaturation of the enzyme occurs during thermocycling. Also, the samplemay include inhibitors of the enzyme requiring isolation of the nucleicacid sample free of inhibiting components. In addition, the methodologyis sensitive to amplifying stray sequences, which then overwhelm thetarget sequence of interest, obscuring its presence. There is also thefact that the reagents are expensive and the amplified DNA usuallyrequires verification. These comments apply equally to the otherenzymatic amplified techniques noted above, such as LCR, NASBA, and SSR.

There is, therefore, substantial interest in identifying alternativetechniques which allow for the detection of specific DNA sequences andavoid the deficiencies of the other systems. Also of interest is thedevelopment of devices for automatically carrying out these alternativenucleotide sequence detection techniques, where these automatic deviceswill reduce the opportunity of error introduction and provide forconsistency of assay conditions.

RELEVANT LITERATURE

Barany, Proc. Natl. Acad. Sci. USA (1991) 88:189-193; Gautelli et al.,Proc. Natl. Acad. Sci. USA (1990) 87:1874-1878. Segev Diagnostics, Inc.WO 90/01069. Imclone Systems, Inc. WO 94/29485. U.S. Pat. Nos.5,185,243, 4,683,202 and 4,683,195.

SUMMARY OF THE INVENTION

Methods and compositions are provided for detecting nucleic acidsequences by using a pair of probes, in each of which at a different endthere is a portion of the chain which serves as one half of a stem,which portion will be referred to as a side chain. The side chainscomprise a cross linking system, which has a photoactivatable entity,normally coupled to a passive reactive entity. Upon orientation of theside chains in spacial proximity as a result of binding of the probes toa contiguous homologous target sequence and activation of the crosslinking system associated with the side chains, the probes are joinedtogether by a covalent linkage. The method employs adding the probes tothe target nucleic acid under conditions of base pairing, activating thecross-linking system, so that primarily only those side chains inspacial proximity form a covalent bond, melting double-stranded nucleicacid and repeating the cycle. Where only one set of probes is used, theexpansion is linear; where complementary sets of probes are used, in there-annealing process the probes in addition to binding to target nucleicacid, will also bind to cross-linked probes. In this manner, one mayobtain a linear or geometric increase in the number of cross-linkedprobes as the cycle of steps is repeated, wherein the process isinitiated by the presence of target DNA.

In a preferred embodiment, the probes have non-cross-linking duplexforming side chains, where at least one side chain is in the form of aduplex prior to hybridization with the target DNA. The side chains arecharacterized that at least one of the side chains has aphotoactivatable group and the other of the side chains has a recipientgroup which reacts with the photoactivatable group to form a covalentbond.

The methods comprise combining the probes whose sequences are homologousto adjacent sequences in the target DNA under conditions, which may besuccessive or simultaneous, which result in melting of the side chainduplexes and hybridization of the probes to the target DNA. Aftersufficient time for hybridization between the probes and the target DNAto occur, the hybridization medium is irradiated to photoactivate thephotoactivatable groups, which will react with the recipient group tocross-link the probes bound to target DNA or dimerized probes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a first embodiment of a control circuit ofan automatic device according to the subject invention;

FIG. 2 is a block diagram for a second embodiment of control circuit ofan automatic device according to the subject invention;

FIG. 3A shows an automatic device according to the subject invention;and

FIG. 3B shows an assay medium unit which is used in conjunction with theautomatic device according to the subject invention.

FIG. 4A shows a first protective system with side chains hybridized and

FIG. 4B shows a first protective system with two probes bound to thetarget.

FIGS. 5 and 6 are diagrammatic views of protective embodiments of thisinvention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for detecting a nucleic acidsequence employing at least one set comprising a pair of first andsecond probes. The pair of probes defines a target sequence, where uponbase pairing of the probes to the target sequence, the probes arebrought into close spacial proximity. Each of the probes has a portionof the probe, which acts as a side chain which does not bind to thetarget sequence. The side chains act as one-half of a stem andnon-covalently interact through hydrogen bonding, salt bridges, and/orVan der Waal forces. When the stem is formed, the side chains comprise acovalent bond cross-linking system, which upon activation results in acovalent bond between the side chains, thus permanently linking theprobes under the conditions of the process.

The method is performed by combining the target nucleic acid with thepair of probes or sets of probe pairs in an appropriate medium for basepairing to produce an assay medium. The nucleic acid may be DNA or RNA,single or double stranded, or other molecule which comprises pyrimidinesand/or purines or their analogs capable of base pairing. Aftersufficient time for the probes to bind to the target nucleic acid or insubsequent steps to bind as well to cross-linked probes, thecross-linking system is activated resulting in covalent bonding betweenthe two probes. One then melts double stranded nucleic acid to releasethe probes from the homologous sequence and repeats the process overagain, whereby the number of cross-linked probes in the presence oftarget sequence is increased linearly or geometrically. Where only oneset of probes is used, linear amplification of cross-linked probes isobtained, which may be satisfactory in many instances.

In describing the subject invention, the probes will be consideredfirst. Each of the probes will have a sequence of at least about 10,more usually at least about 15, preferably at least about 18 and usuallynot more than about 1 kb, more usually not more than about 0.5 kb,preferably in the range of about 18 to 200 nt, and frequently not morethan 60 nucleotides, where the sequence is homologous to the targetsequence. For the most part, the total number of nucleotides which arehomologous to the target sequence for the two probes will be at leastabout 15 nt, more usually at least about 25 nt, and not more than about1.2 kb, usually not more than about 0.5 kb, preferably not more thanabout 300 nt. The base pairing domains present on the target nucleicacid will normally not be separated by more than 10 nt, more usually notmore than about 6 nt, and preferably not more than about 2 nt and may becontiguous.

Desirably, particularly where the side chain is involved with duplexformation (“duplexed side chain”), the probe with the side chain havingthe photoactivatable group will desirably have a fewer number ofcomplementary nucleotides to the target as compared to the probe havingthe recipient group. In this way, where only one probe has hybridized tothe target, it will more likely be the probe with the recipient group,which will not react with the target upon photoactivation.

Each of the probes has a side chain, 3′ on the first probe and 5′ on thesecond probe in the 5′-3′ direction, which will provide for non-covalentassociation to form a stem. Non-covalent association can be obtained byhydrogen bonding, salt bridges, Van der Waal forces, and the like,particularly hydrogen bonding. For the most part, the groups involvedfor association will have oxygen and nitrogen bonded hydrogen, e.g.purines and pyrimidines. Upon activation, covalent cross-linking betweenmembers of the stem occurs. The reaction rate occurring as a result ofthe spacial proximity of the side chains due to the base pairing of theprobes to a homologous sequence will usually be at least about 10 fold,preferably at least about 100 fold, greater than the reaction thatoccurs between the probes unbound to the homologous sequence.

The side chains will be selected so as to have a weak association oraffinity. By weak is intended that in the absence of the target in thesolution, the equilibrium between unassociated probes in solution andassociated probes, due to the affinity between the side chains andtarget homologous nucleic acid sequences will be less than about 10⁻¹,usually less than about 10⁻³ M⁻¹. The affinity may be as a result ofhydrogen bonding, salt formation, or other low energy event.

To obtain stem formation, conveniently, one may use paired nucleotides,at least 2, generally at least 3, and usually not more than about 20,more usually not more than about 16 base pairs, preferably not more thanabout 8 base pairs, more preferably not more than about 6 base pairs,usually in the range of 2 to 6 base pairs, more usually in the range of4 to 6 base pairs. Alternatively, one may use amino acids which providefor hydrogen bonding and/or salt bridges. Other hydrogen bridges mayinvolve diamines and diol acidic groups, particularly ortho-phenolates.However, for the most part, considering convenience, ease of synthesis,control of affinity, and substantial absence of interference,nucleotides, nucleotide analogues or derivatives will be employed, forexample, where the sugars or phosphates may be substituted, base aminoand oxo groups modified, and the like. Usually, the pairs will be A andT, where the nucleotides may be the same on one side chain or different,that is all Ts on one chain and all As on the other chain, or a mixtureof As and Ts on the two side chains. However, one may also use G and C,by themselves or in combination with A and T. Instead of the normal 4 or5 natural bases (including uracil), one may use other bases or othermoieties providing for hydrogen bonding and spacial orientation, such as5-methylcytosine, 5-fluorouracil, 2′-deoxy-5-(trifluoromethyl)uridine,inosine, 1-methylinosine, 3-nitropyrrole, and the like. The particularchoice of nucleotide or substitute moiety will depend on the desiredaffinity, ease of synthesis, interaction with the covalentcross-linking, opportunity to serve as a reactant for cross-linking, andthe like. Generally, the side chains, excluding groups bound to thechain will be at least about 20 atoms in the chain, more usually atleast about 30 atoms in the chain, generally fewer than 100 atoms, moreusually fewer than about 60 atoms. The atoms will be carbon, oxygen,nitrogen, sulfur, phosphorus, and the like. The cross-linking moietiesmay be part of the side chain or appended to the side chain, dependingupon the nature of the moiety.

The base pairing sequences of the two probes will be selected so as toprovide a low affinity between the two probes. Therefore, the targetsequences will be selected so that there will not be a significantnumber of nucleotides defining a sequence of homology, particularlycomplementarity, between the two probes. The greater the complementaritybetween the two probes, the more stringent the conditions will berequired during the period of activation of the cross-linking system.Therefore, one has substantial discretion in the selection of the probesin relation to the conditions employed for base pairing of thehomologous sequences.

The orientation of the stems may be varied, so that the stems may be inthe same or opposite orientation to the target complementary sequence.Thus, one of the stems may be in a parallel orientation to provide forHoogsten base pairing, or both may have anti-parallel orientation, so asto have 3′—3′ coupling of one stem to the target complementary sequenceand analogously 5′—5′ coupling of the other stem to the targetcomplementary sequence.

For geometric expansion, the target complementary portion of the probesneed not, and preferably will not, have target complementary regions ofthe same length. Therefore, when the two complementary probes of the twosets are hybridized, a portion of the target complementary regions willbe exposed generally of from 1 to 10, usually of from about 2 to 6,nucleotides. The exposed portion will be of the 5′ probe in onecombination of probes and the 3′ portion in the other combination. Whenthe four probes are hybridized, all of the complementary regions will behybridized, where a 5′ probe in one combination will extend over the 3′probe of the other combination.

In one embodiment, one of the side chains will provide for a bulgeadjacent to the homologous sequence. The bulge will be between the lastnucleotide base pairing with the target sequence and directly linked tosaid side chain and the first group providing for non-covalentassociation between the side chains to form the stem, e.g. base pairingof nucleotides on respective side chains. Using nucleotides asexemplary, there will usually be 1 to 3 unpaired nucleotides, beforebase pairing occurs between the two side chains. Other groups may beused which provide approximately the same degree of flexibility. Therewill usually be only one bulge, but in some situations, one may have abulge in each side chain.

Both of the members involved in the cross-linking will normally beprovided by an intermediate, at least one of which is not a nucleotideor modified nucleotide, although in some situations one of the membersmay be a nucleotide or modified nucleotide. By employing a difunctionalmolecule for insertion into the chain of the side chain, where thedifunctional molecule carries the cross-linking agent, the members ofthe side chains participating in the cross-linking may be convenientlypositioned for reaction. Various polyfunctional molecules may be used toprovide stable participation of the cross-linking moiety in the sidechain. Desirably, agents will be used which can react with a phosphorusmoiety, particularly a phosphoramidite, or can form a phosphoramidite,where the linking atom may be oxygen, carbon, sulfur, or the like. Coremolecules for linking a cross-linking moiety to the side chain, wherethe core molecule participates in the backbone of the side chain,include glycerol, dithiothreitol, 1,3,5-trihydroxycyclohexane,deoxyribose, 2-hydroxymethylacrylic acid, or the like. Since thephosphorus group can be modified to react with a wide variety offunctionalities, there is no significant restriction on how the coremolecule is fitted into the backbone of the side chain. Phosphorusderivatives include, phosphoramidites, phosphate esters, phosphines,phosphohalides, etc.

In order to reduce the amount of cross-linking of probes in the absenceof being bound to the target molecule, protective systems are provided.The protective system may employ duplex formation, where the duplex maybe solely associated with a side chain, associated with a sequence ofthe probe homologous to the target, or a side chain associated with anadditional molecule. The duplex may form a hairpin (which includes astem and loop), where a hairpin has at least three unmatched contiguousnucleotides. Usually not more than about 8, more usually not more thanabout 5, of the nucleotides are unmatched. By hairpin is intended thatthe turn to form the duplex has at least three nucleotides which areunmatched. By stem and loop is intended that there is more than threeunmatched nucleotides at the turn to form the loop. By a bulge isintended that there are unmatched nucleotides along the stem, whichresults in a bulge. Usually, if there is only one duplex forming sidechain, it will be the side chain with the recipient or passive reactivegroup.

The first protective system has the terminal sequence of one side chaincomplementary in the reverse order, so that the hybridizing sequencesare both in the 5′ - 3′ direction as shown in FIG. 4. The hybridizingsequence of one side chain 11 has a cross-linking group 13 whichcomprises a member of the cross-linking system. In considering how thetwo probes, the 3′ probe 15 and the 5′ probe 17 will exist in solutionif the side chains hybridize, as shown in the FIG. 4A, one shouldpicture the stems forming dsDNA, where the 5′ probe 17 has the member ofthe cross-linking system Y 19 in the hybridizing portion of the sidechain, while the 3′ probe 15 has the member of the cross-linking systemX 13 distal to the hybridizing portion. The vertical lines 21 indicatebase pairing. The 3′ probe 15 is shown as extended so as to hybridize tothe sequence of the 5′ probe complementary to the target, allowing fortriplex formation 23 when the probes are bound to the target 25. Theportion of the stem complementary to the target hybridizing portion willusually not exceed five nucleotides, usually not exceeding threenucleotides. In FIG. 4B, the two probes are bound to the target 25. The3′ probe stem 11 is hybridized to the 5′ probe stem 27, while the 3′probe is hybridized to the target, pulling the linking group around andthe cross-linking member of the 3′ probe 13 into juxtaposition to the 5′probe cross-linking member 19.

By removing a member of the cross-linking system out of the hybridizingregion of the stem, even when the stems are hybridized, the probabilityof obtaining cross-linking without being bound to target issubstantially diminished. Furthermore, by having unreactive groupsopposite the photoactivatable group in the hairpin, uponphotoactivation, there will be no reaction. For example, by using anunreactive group, such as an unsubstituted sugar, dihydrothymidine,pseudouridine, and the like, as the unit across from thephotoactivatable group, the photoactivated group will not have a partnerwith which to react and will return to the ground state from thephotoactivated state to be available for a future reaction with arecipient group.

The linking chain which joins the stem forming sequence to the targethomologous sequence of the probe may comprise any linking system whichdoes not interfere with the purposes of the probe and is convenient froma synthetic standpoint. Desirably, the linking chain is hydrophilic andmay be a polyether, polyester, polypeptide, polyamine, etc. Thus thelinking chain may comprise alkyleneoxy, wherein alkylene will generallybe of from 1 to 3 carbon atoms and the total number of alkylene groupsmay be from 1 to 6, usually 2 to 4, peptide, where the total number ofamino acids will be in the range of 1 to 6, usually 2 to 4, where theamino groups will usually be small, e.g. G and A, or hydrophilic, e.g.S, T, N, Q, D, E, K and R, sugars, where the total number of saccharidicgroups will generally be in the range of 1 to 6, usually 2 to 4, orcombinations thereof, including 1 or more nucleosides which are notinvolved in hybridizing.

Instead of having a hairpin or stem and loop, one may have a bulge whichpreferably includes the photoactivatable group in an unreactiveenvironment. The bulge may be as a result of a hairpin or the additionof an additional sequence partially homologous to the side chain. Forexample, the sequence causing the bulge would lack the passive reactivemoiety, as well as the bases complementary to the bases adjacent to thephotoactivatable group. The bulge causing sequence would have basescomplementary to bases of the side chain distal to the photoactivatablegroup. In the case of a bulge, the side chain will usually have at least6, more usually at least 7 bases, where at least two, preferably atleast three and not more than about 5 bases, will not be matched by thebulge forming sequence. The bases in the bulge may or may not be matchedto provide a sidechain hairpin, the bases usually being other thanthymidine. So long as the bulge forming sequence is bound to the sidechain, the photoactivatable group will be hindered from reacting withthe reciprocal side chain.

Alternatively, one or both of the stems may be extended by anoligonucleotide of from about 2, usually at least 3 to 10, usually notmore than about 8 nucleotides, whose sequence is complementary to aportion of the target complementary portion of the probe sequence. Theduplexing portion would be displaced from the junction of the targetcomplementary sequence to the side chain sequence. This is shown in FIG.5. Again the vertical lines indicate base pairing.

As shown in the figure, the hybridization forms a stem and loop whichincludes the cross-linking member X 41, particularly thephotoactivatable group, so as to create steric hindrance around theportion of the side chain hybridizing with the other side chain. Whereboth the probes have duplexing at their termini, the hybridizing betweenthe two stems will be substantially diminished. However, when the probebinds to the target or a complementary probe, the side chain portionhybridized to the target homologous sequence of the probe will bedisplaced by the target or probe, releasing the side chain to hybridizeto the other side chain to form the stem. The portion of the probe towhich the side chain sequence binds will be selected to bring the sidechain around in a stem and loop, the region beginning not more thanabout 30, usually not more than about 20 nucleotides, from the lastnucleotide hybridizing to the target, and beginning at least about 2,usually at least about 4 nucleotides, from the last nucleotide of theside chain hybridizing to the complementary side chain.

In situations where one has two sets of probes, one may provide for afifth probe or a side chain extension, (hereafter referred to as the“double side chain duplexing sequence”) which serves to hybridize to theside chains on complementary probes. This is exemplified in FIG. 6. Forthe geometric expansion, the complementary probes as pairs 67 and 69 and71 and 73, respectively, may be totally or partially overlapping. Thedouble side chain duplexing sequence 61 would hybridize to the two sidechains 63 and 65 and the available portions of the target complementarysequence 69. The photoreactive or passive groups X, 75 and 77, areshielded, while the passive or photoreactive groups(in relation to thenature of X) 79 and 81, need not be shielded. The double side chainduplexing sequence may include bases which hybridize with portions ofthe probe complementary to the target sequence. The portions willusually not exceed five nucleotides, more usually not exceed fournucleotides, where the double side chain duplexing sequence willdisplace a portion of the sequence homologous to the target. Usually,the double side chain duplexing sequence would have at least about 6members, more usually at least about 7 members and may have up to 30members or more, where there will be complementarity between at least 4members and the side chains, usually at least 5 members and the sidechains, preferably there being at least complementarity between thedouble side chain duplexing sequence and at least 6 nucleotides of theside chains.

There are extensive methodologies for providing cross-linking uponspacial proximity between the side chains of the two probes, to form acovalent bond between one member of the stem and the other member of thestem. Conditions for activation may include photonic, thermal andchemical, although photonic is the primary method, but may be used incombination with the other methods of activation. Therefore, photonicactivation will be primarily discussed as the method of choice, but forcompleteness, alternative methods will be briefly mentioned. In additionto the techniques used to reduce hybridization between the side chainswhen not bound to the target or complementary probe, conditions may alsobe employed to provide for a substantial difference in the reaction ratewhen bound to a template sequence as compared to free in solution. Thiscan be achieved in a wide variety of ways. One can provideconcentrations where events in solution are unlikely and activation ofthe cross-linking group will be sufficiently short lived, so that theactivated group is not likely to encounter another probe in solution.This can be tested using control solutions having known concentrationsof probes and determining the formation of cross-linked probes in thepresence and absence of template. One may use quenchers that act todeactivate cross-linking groups on probes that are free in solution,where the quencher may accept energy, provide a ligand to replace a lostligand, react with the cross-linking group to inhibit cross-linking withanother probe, and the like. By adjusting the amount of quencher in themedium, one can optimize the desired reaction as compared to thebackground reaction. One may use sensitizers, where reaction only occursupon activation of the cross-linking moiety by transfer of energy fromthe sensitizer to the cross-linking moiety. The significant point isthat the sensitizer, which will be bound to the probe carrying thepassive reactive moiety, is directly irradiated and the energy will bedissipated in solution in the absence of the photoactivatablecross-linking moiety accepting the energy. Acceptance of the energy hasa much greater probability when the side chains are involved in stemformation. Sensitizers which may be employed include biphenyl,fluorenone, biacetyl, acetonaphthone, anthraquinone, bibenzoyl, andbenzophenone, or other sensitizers, which because of their tripletenergies, find particular application with the coumarin functionality.These sensitizers may be joined to the side chain in the same manner asthe photoactivatable moiety, as an appropriate site, usually within oneor two bases from the passive reactive moiety.

One can also provide for a substantial difference (between probes boundto a template sequence and probes free in solution) in the reaction rateof the members of the cross-linking system by separating thecross-linking member or activatable member from the sequence providingfor non-covalent association in one of the two side chains of theprobes. In this manner, when the probes are free in solution, althoughthe side chain sequences may be non-covalently associated, uponactivation cross-linking will not occur because the requisite proximityof the cross-linking members of the two side chains will not be present.In contrast, when the probes are bound to a template sequence, e.g. thetarget sequence, the sequences of the side chains will be non-covalentlyassociated and the members of the cross-linking system will also be inthe requisite spacial proximity for activation. The cross-linking memberwill be separated from the sequence in the side chain responsible fornon-covalent association with the side chain of the second probe by asufficient distance so that when the two probes are hybridized to thetemplate sequence, non-covalent association between the side chainsequences may still occur while the activatable members of each sidechain will be in sufficient proximity for activation. Using probes withnucleic acid side chains as exemplary, the separation distance betweenthe sequences responsible for non-covalent association and thecross-linking member of the side chain in the first probe may range from5 to 50 nt, usually from 6 to 40 nt and more usually from 6 to 30 nt.

In one aspect, one can employ photochemistry where a single reactivespecies on one chain reacts with a group present on the second chain. Alarge number of functionalities are photochemically active and can forma covalent bond with almost any organic moiety. These groups includecarbenes, nitrenes, ketenes, free radicals, etc. One can provide for adonor molecule in the bulk solution, so that probes which are not boundto a template will react with the terminating molecule to avoidcross-linking between probes. Carbenes can be obtained from diazocompounds, such as diazonium salts, sulfonylhydrazone salts, ordiaziranes. Ketenes are available from diazoketones or quinone diazides.Nitrenes are available from aryl azides, acyl azides, and azidocompounds. For further information concerning photolytic generation ofan unshared pair of electrons, see A. Schonberg, Preparative OrganicPhotochemistry, Springer-Verlag, NY 1968. Illustrative compounds andterminating molecules include olefins or compounds with a labile proton,e.g. alcohols, amines, etc.

For specificity, one may use a molecule which upon photoactivation formsa covalent bond with a specific other molecule or small group ofmolecules via cycloaddition or photosubstitution reaction. There are asignificant number of compounds which will react with nucleic acid basesto form covalent bonds. Thymidine will react with thymidine to form acovalent link. Preferably, other compounds will be used which react withnucleic acid bases. These compounds will include functional moieties,such as coumarin, as present in substituted coumarins, furocoumarin,isocoumarin, bis-coumarin, psoralen, etc., quinones, pyrones,α,β-unsaturated acids, acid derivatives, e.g. esters, ketones, andnitriles; azido, etc.

Instead of having a reaction with a nucleotide, one can provide for twodifferent reactants, where reaction is unlikely when the two reactantsare not in proximity upon activation. Reactions of this nature includethe Diels-Alder reaction, particularly a photoactivated Diels-Aldercyclization reaction, where a diene, and a dienophile e.g., olefin oracetylene, are employed. Reactive dienes may be employed, such as1,4-diphenylbutadiene, 1,4-dimethylcyclohexadiene, cyclopentadiene,1,1-dimethylcyclopentadiene, butadiene, furan, etc. Dienophiles includemaleimide, indene, phenanthrene, acrylamide, styrene, quinone, etc. Onemay provide for sensitized activation to provide for the cyclization,using such photoactivators as benzophenones with cyclopentadiene, whichmay react with another cyclopentadiene molecule, or a differentdienophile. Alternatively, one may employ addition of ketones toolefins, as in the case of benzophenone and isobutylene or2-cyclohexenone.

Another class of photoactive reactants are organometallic compoundsbased on any of the d- or f-block transition metals. Photoexcitationinduces the loss of a ligand from the metal to provide a vacant siteavailable for substitution. Placement of the organometallic compound onone side chain and a suitable ligand, on the other chain provides asystem which relies on the proximity of the two chains for thecross-linking to occur. Suitable ligands may be the nucleotide itself orother moieties, such as amines, phosphines, isonitriles, alcohols,acids, carbon monoxide, nitrile, etc. For further information regardingthe photosubstitution of organometallic compounds, see “OrganometallicPhotochemistry,” G. L. Geoffrey, M. S. Wrighton, Academic Press, SanFrancisco, Calif., 1979.

By using organometallic compounds having stable coordination complexes,where the ligands can be replaced with other ligands upon photo- orthermal activation, one can provide for stable cross-linking. Examplesof organometallic compounds which may serve as cross-linking agentsinclude four coordinate Group VIII metals, particularly noble metals,cyclopentadienyl metal complexes, having at least one other ligand, andthe like.

One may also employ active monomers which can dimerize with a secondmonomer, such as styrene, acrylonitrile, vinyl acetate, acenaphthylene,anthracene, etc. By activating one of the monomers photolytically, theactivated monomer can react with the other monomer on the other sidechain. Particularly, by using two different monomers, where the secondmonomer provides for a more stable active species than the firstmonomer, one may include a quencher in the reaction medium so as toquench the active intermediate. In some instances, the intermediate willself-quench by elimination or other suitable reaction. One may alsoprovide for photolytically activated homolytic or heterolytic cleavage,such as active halides, e.g. benzyl halides, particularly bromo andiodo, where upon cleavage, the active molecule would act with arecipient molecule, such as an olefin which would provide for additionof the carbon and halogen across the double bond.

Other reactions which might be employed include photonucleophilicaromatic substitution.

Thermal activation may also be employed, but is less desirable in manycases since until the temperature is lowered, the reactive species ismaintained. Therefore, this will usually require lower concentrations ofat least one of the probes, the ability to rapidly change thetemperature of the system, and the selection of reactants which providefor a high energy barrier for reaction in the absence of spacialproximity. Reactions which may be employed include ones described abovefor photolytic activation, such as metal coordination complexcross-linking, and the like. Illustrative of such cross-linking is theuse of platinum tetradentate complexes, e.g. ammonia complexes.

Also, chemical reactions can be employed where one provides for cyclingof the active moiety in the absence of reaction with the recipientreactant. Thus, one can provide for a redox couple, such as ferrous andferric ions, where the active species free in solution would normally beinactivated prior to encountering the recipient compound. For example,one could have a hydroperoxide as the reactant species and an activeolefin as the recipient. Upon reduction of the hydroperoxide, a freeradical can be obtained which can react with the electron donorcompound, which can then be further reduced to a stable compound.

Any of the various groups indicated may be modified by replacement of ahydrogen with a functionality or convenient linking group for attachmentto the backbone of the side chain. These functionalities will, for themost part, be oxy, oxo, amino, thio, and silyl.

The probe homologous sequence which binds to the template will usuallybe naturally occurring nucleotides, but in some instances thephosphate-sugar chain may be modified, by using unnatural sugars, bysubstituting oxygens of the phosphate with sulphur, carbon, nitrogen, orthe like, by modification of the bases, or absence of a base, or othermodification which can provide for synthetic advantages, stability underthe conditions of the assay, resistance to enzymatic degradation, etc.The homologous sequence will usually have fewer than 10 number % of thenucleotides different from the target sequence, and usually the lesserof 10 number % and 10 nucleotides, more usually 5 nucleotides. Therelationship of the pairs of probes will usually come within the samelimitations, but will more usually be complementary, that is, haveperfect nucleotide pairing. Differences between sequences may includeinsertions, deletions, and substitutions, i.e. transitions andtransversions. If one wishes one may have more than one set of a pair ofprobes specific for a target sequence, and may simultaneously have 2 ormore sets of probes, usually not more than 10 different sets, moreusually not more than about 5 different sets, directed to differenttarget sequences. A probe set is one pair for linear expansion and twopairs of probes, for geometric expansion, where for geometric expansion,the probes have homologous binding sequences, so as to bind to targetsequence and to each other. Where one has a plurality of probe sets,each of the probe sets will generally be distinguishable in some assay,for example, by size difference, by label difference, by sequence, etc.

In some instances it may be desirable to provide three different probes,where three probes define three contiguous sequences and two stems, themiddle probe having two side chains, so as to interact with each of theother side chains of the other two probes. This can be particularlyuseful with regions of polymorphism, where the central probe is directedto a conserved region, and one or both of the other probes are directedto polymorphic regions or vice versa. One may then use a plurality ofprobes, one for each of the polymorphic regions, where cross-linkingwill result for any of the polymorphic sequences being present.

The probes may be prepared by any convenient synthetic scheme. In onescheme, the side chains may be prepared first, followed by being linkedto the sequence homologous to the target sequence. The synthesis of theside chains will depend, of course, on the nature of the pairing groups.For oligonucleotides, conventional manual or automated techniques may beemployed. One or more of the monomers may comprise a cross-linkinggroup. By employing a linker in the backbone which employs adeoxyribosylphosphate group or can substitute for thedeoxyribosylphosphate group, the cross-linking containing group may bereadily inserted into the backbone during the synthesis. The side chainsmay have terminal functionalities that allow for direct linkage of thesequence homologous to the target sequence, e.g. a nucleotide5′-triphosphate or nucleotide having a free 3′-hydroxyl. The homologoussequence may be joined by ligation, by using the side chains inconjunction with a primer for PCR, or other linking means. The sidechains may be used to terminate a chain being produced on a bead or maybe the initiating group bound to the bead by a cleavable linker. Thusside chains can be provided as reagents for use in automated synthesis,where the side chains will provide the initiating or terminatingreagent. Various attachment groups may be provided for the side chain,where the side chain is to be attached to a bead. Functionalities on thebead can be hydroxy, carboxy, iminohalide, amino thio, active halogen orpseudohalogen, carbonyl, silyl, etc. For ease of removal from the bead,various linkers may be employed which will include groups, such asbenzhydryl ethers, acetals, including sulfur analogs thereof,o-nitrobenzyl ether, 7-nitroindanyl, cyclic anhydrides, polypeptideshaving a sequence recognized by a peptidase, silanyl, β-(electronwithdrawing group) substituted esters, or the like. The particularlinking group which is selected will depend upon the nature ofcross-linking group and the manner in which it is bonded to the sidechain backbone.

Of particular interest are compositions which provide the hybridizingside chains and can be joined to sequences homologous to targetsequences, to provide probes. Combinations of stem formingoligonucleotides are used. Depending on which technique is used todiminish probe cross-linking background, the compositions providing fora cross-linking member will have the following formula:

N—X_(a)—Z—X_(b)—Z_(c)—(X_(a))_(c)  (1)

N—A—Z—B—X_(a),   (2)

or

N—X¹ _(a)—Z—X² _(b)—A—X³

wherein:

N is a moiety capable of ligation to a nucleotide, which may comprise anhydroxyl group, a phosphate group, a triphosphate group, or the like,including nucleosides, nucleotides, phosphoramidites, phosphate esters,sugars, hydroxyalkyl or -aryl groups, and the like;

X is a nucleotide, naturally occurring or synthetic, capable of hydrogenbonding to another nucleotide, preferably at least one X will beadenosine, and when other than duplex formation of the stem is presentin the probe, usually at least about 50% of the stem base pairing X'swill be adenosine; when Z reacts with thymidine, generally of the totalnucleotides in the stems, at least about 30%, more usually at leastabout 50% will be thymidine and adenosine, where the hybridizingnucleotides have each stem in the same direction, e.g. 5′ - 3′ or 3′ -5or opposite direction, e.g. 5′ - 3′ pairing with 3′ - 5′; thecombination of (1) and (1) and (1) and (3) will have the stemoligonucleotides in the opposite direction, while the combination of (1)and (2) will have the stem oligonucleotides in the same direction;

Z is a cross-linking group having, usually as a side chain, a moietycapable of cross-linking with another moiety, conveniently with anucleotide, or a member of complementary specific reactive pair, moreparticularly as a result of photoactivation (see groups describedabove); or a sensitizer (see groups described above), at least one Z ina combination of stems will be a cross-linking moiety; Z will usually beof at least about 8 atoms other than hydrogen, more usually at leastabout 10 atoms other than hydrogen, and not more than about 50 atoms,more usually not more than about 36 atoms other than hydrogen, where Zmay be aliphatic, alicyclic, aromatic, heterocyclic, or combinationsthereof, where cyclic having from about 1 to 3 rings, which may be fusedor non-fused, composed of carbon, oxygen, nitrogen, sulfur andphosphorus, comprising functional groups, such as oxy, oxo, amino, thio,cyano, nitro, halo, etc., usually having at least one heteroatom, moreusually at least about 3 heteroatoms, and not more than about 10heteroatoms;

X¹ and X² are a nucleotide or oligonucleotide of the stems whichhybridize with each other and will generally be at least 2 nucleotides,having a total number of nucleotides in the range of about 2 to 20,usually 2 to 18, more usually about 3 to 16, and preferably not morethan about 8 hybridizing base pairs, more usually not more than about 6hybridizing base pairs, usually in the range of about 2 to 6, moreusually in the range of 3 to 6, hybridizing base pairs;

X³ is a sequence of at least 2, usually at least 3, nucleotides which iscomplementary to and hybridizes with a sequence of the probe which bindsto the target sequence, so as to form a hairpin comprising at least 3members, usually at least about 4 members and not more than about 12members, usually not more than about 8 members which are not involved inbase pairing, and which hairpin includes Z, where Z will be across-linking member which does not react with the base of a nucleoside;

A and B are linking groups, which will usually be other thannucleotides, where A and B are of sufficient length to permit the twostems to hybridize with each stem in the same direction, e.g. 5′ - 3′ or3′ -5′, so that the number of atoms in A and B will be determined by thelength of the complementary stem, the nature and flexibility of A and B,and the like; usually A and B will have a total of at least about 10atoms in the chain, more usually at least about 12 atoms in the chainand not more than about 60 atoms in the chain, where the side groupswill be selected for synthetic convenience, solubility, inertness,absence of interference in the assay and the like; A and B may bealiphatic, alicyclic, aromatic, heterocyclic or combinations thereof,and may be monomeric or oligomeric, such as polyethers, e.g.polyalkyleneoxy, oligopeptides, e.g. polyglycyl, polyurethanes,polymethylene, e.g. polyethylene, polyacrylate, polyvinylether, etc., ;usually A and B will be at least about 6 carbon atoms, more usually atleast about 8 carbon atoms and not more than 100 carbon atoms, moreusually not more than about 60 carbon atoms and preferably not more than36 carbon atoms, usually having at least 1 heteroatoms, more usually atleast 2 heteroatoms and not more than about 36 heteroatoms, usually notmore than about 20 heteroatoms, where the heteroatoms may be oxygen,nitrogen, sulfur, phosphorus, halogen, and the like; and

a, b and c are integers of a total in the range of 2 to 20, where a isat least one, b may be 0 or greater, usually at least 1, c usually being0 or 1, and the total number of nucleotides for base pairing are atleast 2, usually at least 3 and not more than about 20, usually not morethan about 16, preferably not more than about 8, generally being in therange of 4 to 6 base pairs.

The side chain compositions described above are used in combination forlinking two adjacent sequences homologous to the target sequence. Eitherof the side chain compositions can be selected for linking to the 3′ or5′ terminus of the homologous sequence. The second side chain willusually have nucleotides complementary to the nucleotides of the firstchain to provide hydrogen bonding. In the simplest second chain, it maybe a poly-T, where the cross-linking group reacts with thymidine, andthe nucleotides in the first chain are adenosine. Where the first chainhas other than adenosine bases, the second chain will usually have thecomplementary bases. The first and second side chains can be provided asreagents for linking to the homologous sequences, as termini of primersfor PCR to provide the probes directly, or the like.

In addition, one or both of the side chain compositions may terminatewith a label (including ligand) which allows for detection, such as adirectly detectable label, e.g. radiolabel or fluorescer;chemiluminescer, biotin, antigen, photocatalyst, redox catalyst, or thelike, for detection of the cross-linked probes.

In carrying out the assay, the sample may be subjected to priortreatment. The sample may be a cellular lysate, isolated episomalelement, e.g. YAC, plasmid, etc., virus, purified chromosomal fragments,cDNA generated by reverse transcriptase, mRNA, etc. Depending upon thesource, the nucleic acid may be freed of cellular debris, proteins, DNA,if RNA is of interest, RNA, if DNA is of interest, size selected, gelelectrophoresed, restriction enzyme digested, sheared, fragmented byalkaline hydrolysis, or the like.

For linear expansion, only one pair of probes is required. After eachmelting step, linked probes will be obtained in proportion to the amountof target DNA present. For geometric expansion, two pairs of probes willbe used. Where the target sequence is a single strand, the initial pairwould be homologous to the target and the pair having the analogoussequence to the target added concomitantly or after the first cycle ofcross-linking. Where the sample is double stranded, then both pairs ofprobes, a pair for each strand, are added initially.

The probes and template will be brought together in an appropriatemedium and under conditions which provide for the desired stringency toprovide an assay medium. Therefore, usually buffered solutions will beemployed, employing salts, such as citrate, sodium chloride, tris, EDTA,EGTA, magnesium chloride, etc. See, for example, Molecular Cloning: ALaboratory Manual, eds. Sambrook et al., Cold Spring Harbor Laboratory,Cold Spring Harbor, NY, 1988, for a list of various buffers andconditions, which is not an exhaustive list. Solvents may be water,formamide, DMF, DMSO, HMP, alkanols, and the like, individually or incombination, usually aqueous solvents. Temperatures may range fromambient to elevated temperatures, usually not exceeding about 100° C.,more usually not exceeding about 90° C. Usually, the temperature forphotochemical and chemical cross-linking will be in the range of about20 to 60° C. For thermal cross-linking, the temperature will usually bein the range of about 70 to 120° C.

The ratio of probe to target nucleic acid in the assay medium may bevaried widely, depending upon the nature of the cross-linking agent, thelength of the homology between the probe and the target, the differencesin the nucleotides between the target and the probe, the proportion ofthe target nucleic acid to total nucleic acid, the desired amount ofamplification, or the like. The probes will usually be about at leastequimolar to the target and usually in substantial excess. Generally,the probes will be in at least 10 fold excess, and may be in 10⁶ foldexcess, usually not more than about 10¹² fold excess, more usually notmore than about 10⁹ fold excess in relation to the target during thefirst stage. The initial ratio of probes to target nucleic acid may bemaintained during successive cycles or may be allowed to diminish by theamount of reaction of the reactive species. The ratio of one probe tothe other may also be varied widely, depending upon the nature of theprobes, the differences in length of the homologous sequences, thebinding affinity of the homologous sequences to the target sequence, therole of the probe in the cross-linking system, and the like.Conveniently, the probes may be equimolar, but may vary in the range of1:1-20 more frequently, 1:1-10, where, when there is only one reactiveor activated species, the passive side chain will usually be in excessto substantially ensure that the passive probe is bound to the templatewhenever the photoreactive probe is present on the template.

Where the sample is double stranded, it will usually be denatured, wheredenaturation can be achieved chemically or thermally. Chemicaldenaturation may employ sodium hydroxide in an appropriate bufferedmedium, e.g., tris-EDTA (TE). Triplex formation may be employed.However, where triplex formation requires complexing the probes withRecA, there will generally be no advantage to such a protocol, since itrequires the continuous presence of natural or active RecA which will besubject to denaturation.

During the course of the reaction, depending upon how the assay iscarried out, there may be significant evaporation. Therefore, it willnormally be desirable to put a coating over the assay medium whichinhibits evaporation. Various heavy oils may find use, such as mineraloil, silicone oil, vegetable oil, or the like. Desirably, the oil shouldbe free of any contaminants which might interfere with the assay.Alternatively, one may use sealed systems, where evaporation isinhibited.

The amount of target nucleic acid in the assay medium will generallyrange from about 0.1 yuctomol to about 100 pmol, more usually 1 yuctomolto 10 pmol. The concentration of sample nucleic acid will vary widelydepending on the nature of the sample. Concentrations of sample nucleicacid may vary from about 0.01 μM to 1 μM. In fact, the subject methodhas the capability to detect a single molecule in the absence ofsignificant interference. The amount of the probes may be varied andtheir concentration varied even more widely, in that there will usuallybe at least about an equimolar amount of the probes and as indicatedpreviously, large excesses of one or the other or both of the probes maybe present. Where the target is single stranded, one may initially usesubstantially less of the probe in relation to the target since there isno competition between the probes and an homologous sequence for thetarget. Where the target is double stranded, initially, one willnormally use more of the probes so as to enhance the competitiveadvantage of the probes for the complementary sequences as against thetarget sequences of the sample.

Where chemical denaturation has occurred, normally the medium will beneutralized to allow for hybridization. Various media can be employedfor neutralization, particularly using mild acids and buffers, such asacetic acid, citrate, etc., conveniently in the presence of a smallamount of an innocuous protein, e.g. serum albumin, β-globulin, etc.,generally added to provide a concentration in the range of about 0.5 to2.5%. The particular neutralization buffer employed is selected toprovide the desired stringency for the base pairing during thesubsequent incubation. Conveniently the stringency will employ about1-10× SSC or its equivalent. The base pairing may occur at elevatedtemperature, generally ranging from about 20 to 65° C., more usuallyfrom about 25 to 60° C. The incubation time may be varied widely,depending upon the nature of the sample in the probes, generally beingat least about 5 minutes and not more than 6 hours, more usually atleast about 10 minutes and not more than 2 hours.

After sufficient time for the base pairing to occur, the reactant may beactivated to provide cross-linking. The activation may involve light,heat, chemical reagent, or the like, and will occur through actuation ofan activator, e.g. a means for introducing a chemical agent into themedium, a means for modulating the temperature of the medium, a meansfor irradiating the medium and the like. Where the activatable group isa photoactivatable group, the activator will be an irradiation meanswhere the particular wavelength which is employed may vary from about250 to 650 nm, more usually from about 300 to 450 nm. The intensity willdepend upon the particular reaction and may vary in the range of about0.5 W to 250 W.

In order to obtain amplification, it will now be necessary to meltprobes bound to the template. Melting can be achieved most convenientlyby heat, generally heating to at least about 60° C. and not more thanabout 100° C., generally in the range of about 65° C. to 95° C. for ashort period of time, frequently less than about 5 minutes, usually lessthan about 2 minutes, and normally for at least about 0.1 minute, moreusually for at least about 0.5 minute. While chemical melting may beemployed, it is inefficient and will only be used in specialcircumstances, e.g. thermal activation. After the melting, the mediumwill usually be cooled by at least about 20° C., usually 30° C. or more.During the incubation and photoactivation, the temperature will bedropped to below 65° C., usually below about 55° C. and may be as low as15° C., usually be at least about 40° C.

Activation may then be initiated immediately, or after a shortincubation period, usually less than 1 hour, more usually less than 0.5hour. With photoactivation, usually extended periods of time will beinvolved with the activation, where incubation is also concurrent. Thephotoactivation time will usually be at least about 1 minute and notmore than about 2 hours, more usually at least about 5 minutes and notmore than about 1 hour. This process may be repeated if desired, so thatthe melting-annealing and photoactivation may occur with from 1 to 40cycles, more usually from 1 to 30 cycles, preferably from 1 to 25cycles. During the cycles, the amount of probe may be replenished orenhanced as one proceeds. The enhancement will usually not exceed aboutfive fold, more usually not exceed about two fold.

As the reaction proceeds, in the case of linear expansion, at each stagethere will be hybridization with the target and additional linked probesformed in relation to the amount of target DNA. For geometric expansion,if the original target was single stranded, in the first cross-linkingstep, there will be the target nucleic acid as a template and thecross-linked nucleic acid, which can now serve as a template for theprobes having the same sequence as the target nucleic acid. In the nextstage, one will now produce templates of probes having the same sequenceas the target and the homologous sequence as the target. Thereafter, foreach subsequent cycle, one will form cross-linked probes on the targetsequence template, as well as on the two different cross-linked probetemplates. The situation is analogous with double stranded nucleic acid,except that in the first step one needs to provide probes for bothtarget templates and there is an initial geometrical expansion as toboth of these probe sequences.

The resulting compositions will comprise cross-linked probes. Suchcompositions may be used as probes to identify homologous sequences, toisolate target sequences having homologous sequences, and the like. Thecompositions find articular use in identifying the presence of thetarget sequence in the sample.

At the end of the iterations or cycles of steps, the presence and amountof cross-linked probes may be determined in a variety of ways.Conveniently, gel electrophoresis may be employed and the amount ofcross-linked probes determined by the presence of a radioactive label onone of the probes using autoradiography; by staining the nucleic acidand detecting the amount of dye which binds to the cross-linked probes;by employing an antibody specific for the dimerized probe, particularlythe cross-linked area, so that an immunoassay may be employed; or thelike.

If desired, for quantitation, an internal control may be provided, wherea known amount of a known sequence is introduced, with a known amount ofprobes, equivalent to the probes for the target sequence of interest. Bycarrying out the assay, one would obtain linked probes from the controland linked probes related to any target sequence present in the sample.By taking aliquots of the assay medium during the assay and after eachor different numbers of cycles, one can determine the efficiency of theassay conditions, as well as ratios of cross-linked control probes tocross-linked sample probes. If one has an estimate of the amount ofsample DNA which should be present, one can terminate the assay once theamount of cross-linked control probe indicates that there should besufficient cross-linked sample probe to be detectable. By having afluorescent molecule on one side chain and a quencher molecule on theother side chain, one can monitor the degree of cross-linking inrelation to the change in fluorescence of the assay medium.

Instead of separating the probes from the assay medium, detectiontechniques can be employed which allow for detection during the courseof the assay. For example, each of the probes may be labeled withdifferent fluorophores, where the energy of the emitted light of one ofthe fluorophores is in the absorption band of the other fluorophore. Inthis way, there is only energy transfer when the two fluorophores are inclose proximity. See, for example, U.S. Pat. Nos. 4,174,384, 4,199,599and 4,261,968. By exciting a first fluorophore at a wavelength whichdoes not excite the second fluorophore, where the first fluorophoreemits at a wavelength absorbed by the second fluorophore, one can obtaina large Stokes shift. One reads the fluorescence of the secondfluorophore, which is related to the number of first and secondfluorophores which are in propinquity. During the course of the assay,at the end of each cycle, one can determine the fluorescence of themedium at the emission wavelength of the second fluorophore as a measureof the amount of cross-linking and indicative of the presence of thetarget sequence and its amount. To provide a more quantitativemeasurement, one can use controls having a known amount of targetsequence and compare the fluorescent signals observed with the sampleand control.

By virtue of the fact that one is linking two probes, one can usedifferent labels on the different probes to allow for detection ofcross-linking. Since the two labels will not be held together exceptwhen the two probes are cross-linked, one can use the existence of thetwo labels in a single molecule to measure the cross-linking. Forexample, by having one label which is a member of a specific bindingpair, e.g. antibody and ligand, such as digoxigenin andanti-digoxigenin, biotin and streptavidin, sugars and lectins, etc., andhaving the other label providing a detectable signal either directly orindirectly, one has the opportunity to separate the cross-linked probeson a solid support, e.g. container surface or bead, e.g. magnetic bead,where the detectable label becomes bound to the solid support only whenpart of the cross-linked probes. For direct detection, one may havefluorophores, chemiluminescers, radiolabels, and the like. For indirectdetection, one will usually have a ligand which binds to a reciprocalmember, which in turn is labeled with a detectable label. The detectablelabel may be any of the above labels, as well as an enzyme, where byadding substrate, one can determine the presence of cross-linked probe.

Where one has ternary probes, particularly with a polymorphic target, acentral probe to a conserved region and outer probes for the polymorphicregions, one can use differentially detectable labels on the outerprobes and a ligand on the central probe for separation. In this way,one can readily determine which polymorphism(s) are present. Theseparation of the cross-linked probes provides the advantage ofisolation of the cross-linked probe from the uncross-linked probecarrying the label, allows for washing of the bound probe, and removalof non-specifically bound label. Thus, background due to uncross-linkedlabel can be diminished.

A diverse range of target sequences can be determined in accordance withthe subject protocols. The subject methodology may be used for thedetection of bacterial and viral diseases, plasmid encoded antibioticresistance markers, genetic diseases and genetic testing, veterinaryinfections for commercial livestock and pets, fish stocks in fishfarming, sexing of animals, analysis of water systems for contaminationby organisms or waste materials, and the like.

Among bacterial and viral diseases are: Chlamydia trachomatis, Neisseriagonorrhoeae, Mycobacterium tuberculosis, Haemeophilus ducreyi (chancre,chancroid), Treponema pallidium (syphilis), Helicobacter pylori,Mycoplasma, Pneumocystic carinii, Borrelia burgdorferi (Lyme disease),Salmonella, Legionella, Listeria monocytogenes, HIV I and II, HTLV-II,Hepatitis A, B, C, and D, Cytomegalovirus, human Papillomavirus,Respiratory syncytial virus, Epstein-Barr virus, Dengue (RNA virus),Eastern and Western Encephalitis virus (RNA viruses), Ebola virus, andLassa virus.

Chlamyida trachomatis is the cause of the most prevalent sexuallytransmitted disease in the U.S., leasing to 4 million cases annually.Nucleic acid targets useful for detecting all 15 serovars of C.trachomatis include: 16S ribosomal RNA gene and the rRNA itself, and themajor outer membrane protein (MOMP) gene. C. trachomatis also contains ahighly conserved 7.5 kb cryptic plasmid. Allserovars contain thisplasmid and there are typically 7-10 copies of the plasmid perelementary body.

Neisseria gonorrhoeae, the cause of gonorrhoeae, has species specificsequences useful for its detection, which include: 16S ribosomal RNAgene and the rRNA itself; a 4.2 kb cryptic plasmid that is present in96% of al clinical isolates with approximately 30 copies present in eachbacterium; and the cppB gene, typically present on the plasmid, ispresent in all strains, including those lacking the plasmid.

Mycobacterium tuberculosis, the cause of tuberculosis, has speciesspecific nucleic acid sequences useful for detection, which include: 16Sribosomal RNA gene and the rRNA itself; and an insertion sequence,IS6110, specific for the M. tuberculosis complex, which comprises M.tuberculosis, M. africanum and M. microti. The copy number of theinsertion sequence varies from 1-5 copies in M. bovis to 10-20 copies inM. tuberculosis.

Salmonella has species specific genes which include: an insertionsequence IS200; invAgene, himA gene; and the Salmonella origin ofreplication, on. The invA gene has been identified in 99.4% of about 500strains of Salmonella tested. This gene codes for proteins essential forinvasion by the Salmonella organism into epithelial cells. In addition,142 strains from 21 genera of bacteria different from Salmonella were alfound to lack the invA gene. Similarly, the insertion sequence IS200 hasbeen identified in almost all Salmonella strains. One additionaladvantage in targeting the IS200 sequence is the presence of multiplegene copies in most strains of Salmonella.

Hepatitis B virus is a DNA virus with an unusual genomic organization.Virions are likely to be detected in the blood. There is a high degreeof conservation in many regions of the genome. The genome is small, 3.2kb, and, with overlapping reading frames, there is strong selectionpressure against sequence variation. Candidate probes from the overlapbetween the polymerase and S antigen coding regions include:GTTTTTCTTGTTGAACAAAAATCCT (SEQ ID NO:01)andTTTCTAGGGGGAACACCCGTGTGTCT(SEQ ID NO:02), where the probe would includeat least about 12 nt coming within the indicated sequences.

Hepatitis delta is a single-stranded RNA genome that is encapsulated inHepatitis B virus coat proteins. Delta infection requires simultaneousor pre-existing HBV infection and generally aggravates the clinicalcondition. Virions containing either the delta or HBV genome may bedetected in blood samples. The delta genome encodes one known protein,the delta antigen, that is believed to be required for replicating theviral RNA genome (cellular constituents are also required). Sequences ofinterest as probes come within the sequence:CTGGGAAACATCAAAGGAATTCTCGGAAAGAAAGCCAGCAGTCTCCTCTT TACAGAAAAG (SEQ IDNO:03).

Cytomegalovirus has a large linear double-stranded DNA genome. The virusis found in blood and to a limited extent infects lymphocytes and isalso found in urine. There are repeated regions in the genome allowingfor detection of such repeated regions. Where only limited viraltranscription has occurred, the Immediate Early Region would be thetarget, while for productive infection, probes to the viral glycoproteingenes would be employed.

Human papillomavirus is a circular double-stranded DNA and probes may betargeted to any region of the genome. Of particular interest are probesto the E6/E7 coding region, where one may discriminate betweengenotypes, e.g. HPV 16 and 18, of interest in North America, while othergenotypes, such as 31, 33, 35, 51, and 53 may be diagnostic for cervicalcancer in other parts of the world.

Epstein-Barr virus, the causative agent of mononucleosis and lymphocyticcancers, may be assayed in the sputum.

For acute viral infections, such as Ebola and Lassa, a rapid test notdependent on antibody formation could be of advantage in treating thepatient. CSF fluids may be monitored for bacterial and viral infections,resulting in meningitis and encephalitis. Transplant patients may bemonitored for CMV, herpes, BK and JC viruses.

In the case of plasmid-encoded antibiotic resistance genes, there isgreat concern whether a pathogenic organism is resistant to one or moreantibiotics. Vancomycin is an extremely important drug for treatment ofstrains of Staphylococcus and Streptococcus that are resistant to otherantibiotics. Some strains of enterococcus are resistant to vancomycin.Probing for vancomycin resistance may serve to reduce the transmissionof vancomycin resistance. Probes for detecting vancomycin resistanceinclude:

CATAGGGGATACCAGACAATTCAAAC (SEQ ID NO:04);

ACCTGACCGTGCGCCCTTCACAAAG (SEQ ID NO:05);

ACGATGCCGCCATCCTCCTGCAAAA (SEQ ID NO:06; and (SEQ ID NO:07).

Other targets of interest are the TEM-1 gene (β-lactamase) found inEnterobacteriaceae; TEM-1 gene in penicillinase producing N. gonorrhoeae(PPNG) plasmid; genes conferring aminoglycoside antibiotic resistance;genes conferring erythromycin resistance; and genes conferring rifampinresistance, especially associated with M. tuberculosis.

Also of interest is amniocentesis or other procedure for isolating fetalDNA, where the interest may be in the sex of the fetus, grosschromosomal aberrations, e.g Down's syndrome, where one would quantitatethe level of chromosome 21.

The sequences specific for the various pathogens, genes or the like willprovide for specificity as to a particular genus, species, strain, or aparticular gene, structural or non-structural. Usually, at least 15,more usually at least 18 nt probes will be employed which are homologousto the target of interest. These homologous sequences are joined to anappropriate side chain to provide the probe. There will be at least oneset of probes, usually at least two sets of probes, where the two setsare homologous to complementary strands of the target sequence.Combinations of sets of probes for the pathogens may be provided askits, where more than one portion of the target host genome may betargeted for binding by the probes. Probes will be selected to providefor minimum false positives, screening the probes with samples from aplurality of individuals from whom one would obtain physiologicalsamples, e.g. blood, serum, urine, spinal fluid, saliva, sweat, hair, orother source of DNA or RNA to be detected.

The samples will be processed in accordance with conventional ways.Where cells are involved, the cells may be lysed chemically ormechanically and the nucleic acid isolated. Where RNA is the target,inhibitors of RNAses will be employed and the RNA will usually bereverse transcribed to provide the target sequence as DNA. Processingmay involve fragmentation of the nucleic acid by mechanical means,restriction enzymes, etc. Separations may be involved, where the nucleicacid may be separated by size, e.g. electrophoresis, chromatography,sedimentation, etc. Usually, the nucleic acid will be freed of othercomponents of the lysate, such as membranes, proteins, sugars, etc.,frequently being denatured in the process. The particular manner ofisolating the target nucleic acid is not critical and will be chosen inaccordance with the nature of the sample, the nature of the target, andthe like.

For carrying out the methodology, various heating and cooling systemsmay be employed, such as a thermal cycler, regulated temperature baths,and the like.

The repetitive nature of some of the steps of the methodology, e.g.melting and annealing of nucleotide sequences and activation of theactivatable groups of the probes, provides for the opportunity ofemploying automatic devices for performing the subject assays. Ofinterest are automatic devices which automate the (1) preincubation, (2)hybridization, (3) photoirradiation, (4) denaturation and (5)post-processing steps of the subject methodology, and which are capableof cycling between steps 2-4. Automatic devices which may be employedwill generally comprise a means for controlling the base pairing orhybridization conditions of the assay medium, e.g. for modulating thetemperature of the medium; and a means for actuating, in a mannerresponsive to the conditions of the assay medium, an activator of theactivatable groups of the probes.

The means for controlling the base pairing conditions of the assaymedium may be any means capable of modulating the conditions of themedium, preferably reversibly, from a first state in which base pairingof complementary nucleotide sequences occurs, i.e. medium conditionsconducive to annealing or hybridization of complementary nucleotidesequences, to a second state in which base-paired or hybridizednucleotide sequences dissociate or melt. As described above, theconditions of the assay medium may be modulated through both thermal andchemical means, but thermal means are preferred. Thus, the means may beone which is capable of reversibly modulating these conditions of theassay medium.

Where melting and annealing of complementary nucleotide strands duringan assay is accomplished through changes in the thermal conditions ofthe medium, the means for modulating the base pairing conditions will beone which is capable of changing the temperature of the medium from afirst temperature in a range at which base pairing occurs to a secondtemperature in a range at which annealed nucleotide sequencesdissociate. The thermal modulation means should be able to maintain theassay medium at a substantially constant temperature, i.e. within a 1 to2° C. variation, within the ranges of the first and second temperatures.Furthermore, the thermal modulation means will preferably provide for anadjustable rate of transition between the first and second temperatures.Suitable means for thermal modulation of the assay medium includethermal cyclers, and the like.

Also present in the subject devices will be a means for actuating anactivator of the activatable groups of the probes. This actuating meansis responsive to assay medium conditions, so that the activator of thecross-linking system, e.g. the source of irradiation in photoactivatablesystems, is operative during conditions of base pairing and inoperativeduring conditions of nucleotide dissociation or melting. Conveniently,this activation means may be a circuit that is configured to beresponsive to the assay medium conditions and controls the operation ofthe activator.

Control circuits which may be employed in the subject devices arecircuits configured to actuate an activator, e.g. an irradiation means,at a predetermined assay medium condition or set of assay mediumconditions. Suitable control circuits will include a means fortransducing the conditions of the assay medium into an electrical signaland a means for triggering the activator in response to the transducedelectrical signal. Illustrative control circuits which may be employedin the subject devices are provided in FIGS. 1 and 2.

FIG. 1 provides a block diagram of a control circuit where anirradiation source, the activator, is activated when the temperature ofthe assay medium is below a predetermined temperature or settemperature, e.g. below the temperature at which base pairing ofcomplementary nucleotide sequences occurs. Circuit 10 comprises athermistor 12 whose resistance varies in response to changes in thetemperature of the assay medium with which it is in contact. Circuit 10also comprises a potentiometer or variable resistor 14, an operationalor differential amplifier 16 and a transistor 18 which collectivelyoperate to activate irradiation source 20 via switch or relay 22 whenthe temperature of the medium is below the set temperature. Circuit 10also comprises LED 24 which signals that switch 26 is closed, therebyclosing the circuit loop.

During operation, the set temperature of the assay medium below whichthe circuit will actuate the irradiation source is controlled byadjusting potentiometer 14. When the temperature measured by thermistor12 is above the set temperature, the resistance of the thermistordecreases so that the output of operational amplifier 16 is insufficientto activate the transistor 18. Since the transistor 18 is inactive,current does not flow through relay 28 and light circuit 22 remains inthe open position, whereby the irradiation source remains inactive. Whenthe temperature sensed by thermistor drops below the set temperature,the resistance of the thermistor increases to a point at which theoutput of operational amplifier 16 is sufficient to activate transistor18. Since the transistor 8 is now activated, current flows through relay28 and light circuit 22 closes (not shown), whereby the irradiationsource is turned on.

Instead of having a circuit which is responsive to a single assay mediumcondition, e.g. a single temperature, circuits responsive to a set ofassay medium conditions, such as two temperatures, may be successfullyemployed. FIG. 2 provides a block diagram of a second control circuitwherein the irradiation source is only activated when the temperature ofthe assay medium is within a narrow, predetermined temperature range,e.g. between 40 and 43° C. In other words, the irradiation source isactivated when the temperature of the assay medium is: (a) below a firstpredetermined or set temperature and (b) above a second predetermined orset temperature. In FIG. 2, circuit 40 comprises a first loop 42 whichis analogous to circuit 10 in FIG. 1 and a second loop 44 which isparallel with first loop 42, where second loop 44 comprises a secondoperational amplifier 46 and transistor 48. As in the circuit depictedin FIG. 1, the output of operational amplifier 58 is only sufficient toactivate transistor 60 and thereby close light circuit 62 via activationof switch 66 when the temperature of the assay medium sensed bythermistor 50 is below a first set temperature T1. The first settemperature T1 is determined by potentiometer 64. The output ofoperational amplifier 46 is sufficient to activate transistor 48 onlywhen the temperature of the assay medium, as sensed by thermistor 50,exceeds a set temperature T2, a fixed temperature below T1. T2 isdetermined by resistors 52, 54 and 56, where the choice of resistancevalues may be readily determined by calculation depending on the desiredset temperature T2. Since both transistors 60 and 48 must be activatedfor current to flow through relay 66, light circuit 62 will only beclosed, thereby activating irradiation source 20, when the temperatureof the assay medium as determined by thermistor 50 is between T1 and T2.

Automatic devices according to the subject invention will also comprisean assay containment means for holding the assay medium during theassay. The assay containment means may be any means capable of holding afixed volume of assay medium, where the containment means will allow formodulation of the base pairing conditions of the medium and activationof the activatable groups by the activator of the device. For example,where a thermal modulation means is employed, the containment meansshould allow for accurate temperature control of the medium in thecontainment means, e.g. an eppendorf tube in a thermal cycler. Whereactivation is accomplished by irradiation, the containment means shouldallow for irradiation of the sample, where the shape of the containmentmeans may provide for substantially uniform irradiation of the sample,e.g. a container which holds the assay medium in thin, film like layer.The containment means may be any convenient shape, such as a vial, tube,slide, channel, chamber, cylinder and the like.

Automatic devices according to the subject invention comprising meansfor modulating the base pairing conditions of the assay medium and meansfor actuating an activator in a manner responsive to the assayconditions may conveniently be housed in a housing, where the housingcomprises means for controlling and/or adjusting the various elements ofthe device, such as on-off switches, toggle switches, dials and thelike.

An automatic device for performing the subject assay which incorporatesa control circuit as described above is shown in FIG. 3. In FIG. 3,device 70 comprises thermocycler 72 and control box 80. Positioned overthe sample holder (not shown) of the thermal cycler 72 is light bank 76with which the assay medium in the sample holder shown in FIG. 3B is inlight receiving relationship. Control box 80 is in electricalcommunication with thermocycler 72 via leads 78.

Control box 80 comprises dial 82 that adjusts the set temperature of thecontrol circuit at which the light bank is activated by adjusting thepotentiometer of the circuit. The toggle main switch 89 turns thecontrol box on, as indicated by red LED 88, while push button switch 88closes and activates the control circuit loop of the subject device, asindicated by illumination of green LED 86.

In FIG. 3B, assay medium unit 90, which is placed within thethermocycler 72 and is in light receiving relationship with light bank76, comprises a tube holder 92 and an eppendorf tube or microtiter platewell 94 comprising the assay medium. Immersed in the assay medium isthermistor 98 which is in electrical communication with the controlcircuit of the device via leads 96.

Kits are provided having at least two pairs of probes, or ternarycombinations of probes, where each pair may be in the same vessel. Atleast one pair will define a substantially contiguous sequence of atarget nucleic acid and the other pair will be homologous, usuallycomplementary, to the sequence of the first pair. Each probe has a sidechain which forms a stem with the side chain of the other pair, so as tobe capable of cross-linking as described previously. If desired, one orboth of the probes may be labeled to allow for easy detection ofcross-linked probes. One may use radioactive labels, fluorescent labels,specific binding pair member labels, and the like. The kits may haveoligonucleotides which include sequences for hybridizing to a targetnucleic acid or provide only the side chains for linking to such targethomologous sequences. For the side chain sequences, these will have atleast two nucleotides in addition to the cross-linking entity andusually not more than about 150, more usually not more than about 100,usually not more than about 60, depending upon whether a protectivegroup is present. If the protective group is not present, the side chainby itself will usually not exceed 20 nucleotides, more usually notexceed about 12 nucleotides. The terminal nucleotide may befunctionalized appropriately for linking to the target homologoussequence.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Preparation of the Photocrosslinker Reagent1-O-(4,4′-Dimethoxytrityl)-3-O-7-coumarinyl)-2-O-(β-cyanoethyl-N,N-diisopropylphosphoramidite) glycerol.

The title compound, prepared in four steps starting from7-hydroxycoumarin, is useful for incorporating the photocrosslinker intoany position in the sequence of an oligonucleotide via automatedsynthesis. Synthesis of 7-glycidyl coumarin: To 270 mL acetone in areaction flask equipped with a reflux condenser was added7-hydroxycoumarin (16.2 g), epibromohydrin (15.8 g) and potassiumcarbonate (13.8 g) and the mixture was refluxed for 18 h. After coolingthe reaction mixture, 100 mL 5% sodium hydroxide (aqueous) was added andthe solution was extracted three times with 80 mL methylene chloride.The extracts were combined and the solvent removed by rotary evaporationto give the crude product as a yellow solid (1.5 g). The product waspurified by recrystallization from hexane: acetone (3:2) at 4° C. toafford a white powder (290 mg): mp 110-112° C.; TLC (8% v/v ethylacetate/chloroform) R_(f)=0.6.

Synthesis of 1-O-(7-coumarinyl) glycerol: 7-Glycidyl coumarin (2.0 g)was dissolved in 80 mL acetone and 50 mL 1.8 M sulfuric acid, and thesolution was refluxed for 20 minutes. The solution was cooled to roomtemperature, neutralized with 1.6 M ammonium hydroxide, and extractedthree times with 50 mL ethyl acetate. The combined extracts wereevaporated to yield the product as a white solid (1.40 g): mp 118-120°C.

Synthesis of 1-O-(4,4′-Dimethoxytrityl)-3-O-(7-coumarinyl) glycerol: Thestarting material 1-O-(7-coumarinyl) glycerol (1.37 g) was dried bycoevaporation with 11 mL pyridine, repeated three times. To the driedmaterial was added 45 mL pyridine, 0.33 mL triethylamine,4-dimethylaminopyridine (44 mg) and dimethoxytrityl chloride (1.78 g).The solution was stirred at room temperature for 3 h, 66 mL water wasadded, and the solution was extracted three times with 35 mL methylenechloride. The organic extract was dried with sodium sulfate and thesolvent was removed to give the crude product. Purification by silicagel column chromatography using hexane:acetone:triethylamine (70:28:2)yielded the product as a white solid (2.6 g): TLC (same solvent)R_(f)=0.43.

Synthesis of1-O-(4,4′-Dimethoxytrityl)-3-O-(7-coumarinyl)-2-O-(β-cyanoethyl-N,N-diisopropylphosphoramidite) glycerol: The starting material1-O-(4,4′-Dimethoxytrityl)-3-O-(7-coumarinyl) glycerol was dried bycoevaporation with 12 mL pyridine: chloroform (3:1), repeated twice. Theresulting viscous liquid was dissolved in 10 mL pyridine: chloroform(1:1) and added under argon with rapid stirring to a flask containing 10mL methylene chloride, 3 mL N,N-diisopropylethylamine, andβ-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (1.8 g). The solutionwas stirred for 90 minutes. The solution was diluted with 60 mL ethylacetate and 3 mL triethylamine, then washed twice with 50 mL saturatedaqueous sodium chloride. The organic phase was dried with sodium sulfateand the solvent was removed to give the crude product. Purification bysilica gel column chromatography using hexane:acetone:triethylamine(70:28:2) yielded the product as a viscous, clear oil (2.6 g): TLC(hexane:acetone, 4:1) R_(f)=0.20.

Oligonucleotide synthesis: For use in automated oligonucleotidesynthesis, the photocrosslinking reagent was dissolved in dryacetonitrile at a concentration of 0.5 g/mL. The bottle of the solutionwas affixed to an extra port on the synthesizer and incorporated via thepreprogrammed protocol. After automated synthesis, the oligonucleotidewas cleaved from the solid support and deprotected with 3 mL 30%ammonium hydroxide for 2 h at room temperature. The ammonium hydroxidewas removed in vacuo, and the oligonucleotide was purified tohomogeneity by denaturing polyacrylamide gel electrophoresis. Stocksolutions in distilled, de-ionized water were prepared and stored untiluse at −20° C.

Sequences of nucleic acids employed in Examples 1 & 2

Nax 228 (SEQ ID NO:08) 5′ATTTTGTCTTTGCGCACAGACGATCTATTT3′

Nax 229 (SEQ ID NO:09) 3′TTTCGTTTGTCTGCTAGATAAA5′

Nax 230 (SEQ ID NO: 10) 3′TAAAACAGAAACGCGCGAXA5′

Nax 231(SEQ ID NO:11) 5′ATTTTGTCTTTGCGCGGCTTT3′

Nax 232(SEQ ID NO: 12) 3′AXACGTTTGTCTGCTAGATAAA5′

Nax 233 (SEQ ID NO: 13) 3′TAAAACAGAAACGCGCGTTT5′

X=ethoxycoumarin 1. The ability to obtain cross-linking with aphotoactivatable probe was investigated.

Component, pm/ Sample, μl Nax μl* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16³²P-228 0.5  2  2  2 2  2  2  2  2 ³²P-229 ″  2  2  2 2 ³²P-233 ″  4 4 44 228 ″  2 2 2 2 229 ″  2 2 230 ″  2 2  2 2 232 ″  2 2 2 2 233 ″  2 2H₂O 12 10 10 8 12 10 10 8 12 10 10 8 10 8 8 6 *pmol/μl Total volume =32.5 μl

Protocol

Add 18.5 μ; of 50:150 0.75M NaOH: 1×TE to 14 μl of sample.

Incubate at room temperature for 10 minutes.

Add 17.5 μl neutralization buffer: 3.5 μl of 3.5% BSA; 1.5 μl of 1.5MHOAc; 11.3 μl of 20×SSC and 0.4 μl of water.

Incubate at 40° C. for 15 minutes.

Irradiate at 30° C. for 1 hour (Stratalinker; thin pyrex filter) PAGE15% (with 7M urea)

The results of the PAGE showed that samples 3, 8 and 10 showed goodcross-linking, but the band for sample 16 was light as compared to theother bands.

2. The effect of thermal cycling on cross-linking was investigated.

pm/ Sample, μl Component, Nax μl* 1 2 3 4 5 6 7 8 ³²P-229 1  1  1  1  1³²P-233 ″  1  1  1  1 228 0.02  1  1  1  1 230 0.5  2  2  2  2 232 ″  2 2  2  2 H₂O 11 10 11 10 11 10 11 10 *pmol/μl

Protocol

Add 18.5 μl of 50:150 0.75M NaOH: 1×TE to 14 μl of sample.

Incubate at room temperature for 10 minutes.

Add 17.5 μl neutralization buffer: 3.5 μl of 3.5% BSA; 1.5 μl of 1.5MHOAc; 11.3 μl of 20×SSC and 0.4 μl of water.

Incubate at 40° C. for 15 minutes.

Irradiate at 40° C. for 25 minutes (Stratalinker; thin pyrex filter)Remove samples, 3,4,7,8, as before; heat to 88° C. for 1 minute.

Cycle:

Irradiate at 30° C. for 25 minutes.

Remove samples, heat to 88° C. for 1 minute.

Repeat cycle 3 times ending with irradiation

PAGE 17% (with 7M urea)

Based on the PAGE results, samples 1, 3, 5, and 7 showed that with orwithout thermocycling, in the absence of the target strand, the twoprobes do not significantly cross-link. Cross-linking was more efficientwith probes 229 and 230. The extent of cross-linking was quantified forsamples 2 and 4, where cross-linking was 2.3% and 7.8% respectively.

Sequences of Nucleic acids use in Examples 3-6:

Nax 238 (SEQ ID NO: 14)

5′TTTATAAAAAGCTCGTAATATGCAAGAGCATTGTAAGCAGAAGACTTA3′

Nax 271 (SEQ ID NO: 15) 5′TTTATAAAAAGCTCGTAATATGCTTTTTTTTT3′

Nax 270 (SEQ ID NO: 16) 3′TTTTTTTTTCTCGTAACATTCGTCTTCTGAAT5′

Nax 272 (SEQ ID NO:18) 3′AAATATTTTTCGAGCATTATACGAXA5′

Nax 273 (SEQ ID NO: 19) 3′AAATATTTTTCGAGCATTATACGAAAXA5′

Nax 274 (SEQ ID NO:20) 3′AAATATTTTTCGAGCATTATACGAAXAAAA5′

Nax 275 (SEQ ID NO:21) 3′AAATATTTTTCGAGCATTATACGAAAAAXA5′

Nax 239 (SEQ ID NO:22)3′AAATATTTTTCGAGCATTATACGTTCTCGTAACATTCGTCTTCTGAAT5′

Nax 278 (SEQ ID NO:23)3′TAAATATTTTTCGAGCATTATACGTTCAAGTAACATTCGTCTTCTGAAT5′

Nax 277 (SEQ ID NO:24) 3′AAATATTTTTCGAGCATTATACGTTCTTTTTTTTT5′

Nax 276 (SEQ ID NO:25) 5′TTTTTTTTTCATTGTAAGCAGAAGACTTA3′

Nax 279 (SEQ ID NO:26) 5′TTTATAAAAAGCTCGTAATATGCAAGAAXAAAA3′

Nax 280 (SEQ ID NO:27) 5′TTTATAAAAAGCTCGTAATATGCAAGAXAAAAA3′

3. The effect of having the reactive group at the 5′ terminus wasinvestigated.

pm/ Sample, μl Component, Nax μl* 1 2 3 4 5 6 7 8 9 10 11 12 ³²P-270 0.5 2 2  2 2  2 2 238 ″ 1 1 1 1 271 ″  2  2  2  2 272 ″  1  1 1 273 ″  1  11 274 ″  1  1 1 275 ″  1  1 1 H₂O 11 11 9 11 11 9 11 11 9 11 11 9*pmol/μl

Protocol

Add 18.5 μl of 50:150 0.75M NaOH: 1×TE to 14 μl of sample into 96 wellCoStar.

Incubate at room temperature for 10 minutes.

Add 17.5 μl neutralization buffer: 3.5 μl of 3.5% BSA; 1.5 μl of 1.5MHOAc; 11.3 μl of 20×SSC and 0.4 μl of water.

Add 75 μl mineral oil to inhibit evaporation.

Incubate at 40° C. for 20 minutes.

Irradiate at 40° C. for 20 minutes (UV-A lamp, UV-32 Hoya filter)

PAGE 20% with 7M urea.

The percent cross-linking with the reactive entity at the 5′ terminuswas: 1, 80%; 3, 69%; 4, 57%; 6, 69%; 7, 68%; 9, 80%; 10, 38%; and 12,67%. There was no significant cross-linking observed where there was notemplate.

4. The effect of having the reactive group at the 3′ terminus wasinvestigated.

Sample, μl

pm/ Component, Nax μl* 1 2 3 4 5 6 7 8 ³²P-276 0.5  2  2  2  2  2  2³²P-277 ″  2  2 239 ″  1  1 278 ″  1  1 279 ″  1  1  1  1 280 ″  1  1  1 1 H₂O 11 11 10 10 11 11 10 10 *pmol/μl

The protocol was the same as the previous example, except that the PAGEwas 18%.

The percent cross-linking with the reactive entity at the 3′ terminuswas: 1, 86; 3, 73%; 4, 83%; 5, 79%; 7, 42%; and 8, 77%. There was nosignificant cross-linking observed where there was no template.

5. The time dependency of cross-linking efficiency was determined.

pm/ Component, Nax μl* 1 2 3 4 5 6 7 8 ³²P-270 0.5  2  2  2  2 ³²P-276 ″ 2  2  2  2 238 5    1  1  1  1 274 ″  1  1  1  1 278 ″  1  1  1  1 279″  1  1  1  1 H₂O 10 10 10 10 10 10 10 10 *pmol/μl

Protocol

The above protocol was followed to the incubation at 40° C. for 15minutes, where irradiation was then carried out for 20 minutes, withsamples 1 and 2 being removed after 5 minutes, 3 and 4 after the next 5minutes, and so on, followed by PAGE 20% with 7M urea.

The percent cross-linking observed was: sample 1, 65%; 2, 72%; 3, 76%;4, 80%; 5, 80%; 6, 83%; 7, 82%; and 8, 84%. The odd-numbered samples hadthe reactive group on the 5′ terminus, while the even numbered sampleshad the reactive group on the 3′ terminus. The results indicate thatafter 10 minutes there does not seem to be any change in the degree ofcross-linking and that there is no significant difference in result,whether the reactive group is on the 5′ or 3′ terminus.

6. The effect of variation in concentration of the probes wasinvestigated.

pm/ Samples, μl Component, Nax μl* 1 2 3 4 5 6 7 8 ³²P-276 0.5  2 2 2 11 1 1 1 278**  1 2 2 2 1 5 2.5 1 279 0.5  2 2 2 2 2 2 2 2 H₂O 10 8 8 910  6 8.5 10  *pmol/μl **278 was 5 pmol/μl for sample 1, 0.5 pmol/μl forsamples 2-5, and 0.02 pmol/μl for samples 6 to 8.

Protocol

The sample was prepared as previously described, followed by incubationat 40° C. for 10 minutes. Samples 1 and 2 were removed from the plateand put in Robbins Scientific PCR tubes (clear) and capped. The PCRtubes were laid across the top of a 96-well plate and irradiated 20minutes (UV-A, UV-32). The samples were analyzed with PAGE 20% with 7Murea.

The degree of cross-linking observed in the samples was as follows:sample 1, 83%; 2, 81%; 3, 79%, 4, 82%; 5, 78%; 6, 17%; 7, 8.2%; and3.9%. At 0.1 pmol of the probe, the degree of cross-linking hassignificantly diminished, but even at 0.05 pmol, cross-linking is stilldiscernible. The effect results from a combination of a lowerconcentration of the probe and lower mole ratio of the probe totemplate.

7. Use of Cross-linked Probes as a Template was Investigated.

Cross-linked products were prepared on a preparative scale and isolatedand purified using PAGE. The five cross-linked products were 345-346,386-346, 387 346, 388-346, and 389-346.

Nucleic Acid Sequences used in Example 7.

NAX 342 (SEQ ID NO:27 )5′-GATATCGGATTTACCAAATACGGCGGGCCCGCCGTTAGCTAACGCTAATCGATT

NAX 345 (SEQ ID NO: 28 ) 5′-AAAAAXAGCCGTTAGCTAACGCTAATCGATT

NAX 346 (SEQ ID NO: 29) 5′-GATATCGGATTTACCAAATACGGCGGGCCCTTTTTTT

NAX 347 (SEQ ID NO: 30) 5′-AAAAAXAGCCGTATTTGGTAAATCCGATATC

NAX 348 (SEQ ID NO: 31) 5′-AATCGATTAGCGTTAGCTAACGGCGGGCCCTTTTTTT

NAX 386 (SEQ ID NO: 32) 5′-AAAXAAGCCGTTAGCTAACGCTAATCGATT

NAX 387 (SEQ ID NO: 33) 5′-AAXAAAGCCGTTAGCTAACGCTAATCGATT

NAX 388 (SEQ ID NO: 34) 5′-AXAAAAGCCGTTAGCTAACGCTAATCGATT

NAX 389 (SEQ ID NO: 35 ) 5′-XAAAAAGCCGTTAGCTAACGCTAATCGATT

Component, pmol/m Sample [mL] NAX L 1 2 3 4 5 6 ³²P-348 0.5 1 1 1 1 1 1342 5 1 345-346 2.5 2 386-346 ″ 2 387-346 ″ 2 388-346 ″ 2 389-346 ″ 2347 5 1 1 1 1 1 1 H₂O 11  10  10  10  10  10 

Protocol

The samples were prepared as previously described, except only 70 mL ofmineral oil was employed. The samples were incubated at 40° C. for 20minutes. The samples were then irradiated at 40° C. for 20 minutes,followed by analysis by PAGE, 17% polyacrylamide and 7 M urea.

The percent cross-linking as a result of the cross-linked probes actingas a template in comparison with a single-stranded template is asfollows: sample 1, 73%; 2, 75%; 3, 71%; 4, 69%; 5, 66%; and 6, 67%. Theresults demonstrate that the cross-linked probes can serve as a templatefor cross-linking a hybridized probe pair as effectively as asingle-stranded target can serve as a template.

8. Linear amplification is demonstrated in the following twoexemplifications.

Example A.

pm/λ Samples, μl Component, Nax * 1 2 3 4 5 6 7 8 ³²P-276 0.5 1 1 1 1 11 1 1 278 .005 1 1 1 1 1 1 1 # 279 0.5 2 2 2 2 2 2 2 2 H₂O 1 1 1 1 1 1 11 0 0 0 0 0 0 0 1 Conditions No. irradiations 1 5 5 1 1 1 1 1 0 0 5 5 5Heat treat- ment — Δ + Δ + Δ + + *pmol/λ; # add 0.2 λ of 0.5 pmol/λafter 10 irradiations Δ heat cycle set forth below; + isothermal

Protocol

The samples were prepared as previously described, with the probes at100-fold excess over the target sequence.

The reagents were combined in 0.2 ml PCR tubes from MJ Research andcovered with 60 μl mineral oil.

All incubations were done on a PTC-100 thermal controller from MJResearch.

The assay mixture was incubated at 40° C. for 15 minutes.

Irradiation was for 15 minutes (Autoprobe, 40° C., UV-A, UV-32).

Samples 2, 4, and 6 were treated in PTC-100 (Program name PCA 8640, 4minutes at 86° C.; 11 minutes at 40° C.) Samples 3, 5, 7, 8 were left atroom temperature.

The irradiation was repeated, with samples 2, 3 being removed after 5irradiations, cycling continued, but with the following schedule:irradiation time: 5 minutes; heating time: 2 minutes at 86° C.;incubation time: S minutes at 40° C.

Some cloudiness was observed in samples 4 and 6 after the 6th cycle. Theheating temperature was reduced to 82° C. for the 7th heating cycle.

PAGE 17% 7M urea.

The following table indicates the results.

% cross- Total # Sample linked Counts Unreact-ed Cross-linked Cycles 11.2 11754  11607  147  1 2 6.6 7027 6563 464  5 3 2.8 8272 8037 235 ″ 48.0 7094 6528 566 10 5 2.9 7953 7722 231 ″ 6 9.4 7280 6595 685 15 7 4.07020 6734 286 ″ 8 23 7000 5418 1582  ″

Example B.

Samples, μl Component, Nax pm/λ* 1 2 3 4 5 6 7 8 ³²P-276 0.5 2 2 2 2 2 22 278 .02 .8 .8 .8 .8 .8 .8 .8 .8 279 0.5 2.5 2.5 2.5 2.5 2.5 2.5 2.52.5 H₂O 9 9 9 9 9 9 9 9 Conditions No. irradiations 1 2 10 10 10 10 1010 Heat treatment − Δ Δ Δ Δ + + + *pmol/λ; Δ heat cycle set forthbelow; + isothermal

Protocol

The above procedure was repeated with some modifications. The probe wasin 50-fold excess to the target. 75 μl of mineral oil was used. Thereactions were run in a polycarbonate plate. Incubation and heating wereon a MJ Research PTC-100 instrument. Irradiation was in a Stratalinkerwith the heating provided by a mineral oil bath set at 40° C.

Sample 1 was removed after one cycle of irradiation and heating; sample2 was removed after one cycle of irradiation, heating and an additionalirradiation. Samples 3, 4 and 5 received 10 cycles of irradiation of 10minutes each, with 9 intervening thermal denaturation cycles inaccordance with the following schedule: 84° C. for 3 minutes; 40° C. for7 minutes. Samples 6, 7 and 8 received 10 cycles of irradiation with 9intervening cycles of remaining in the mineral bath inside theStratalinker. The following table indicates the results.

% Cross- Total # Sample linked Counts Unreacted Cross-linked Cycles 11.6 11641 11458 183  1 2 2.2 16744 16381 363 10 3 11.7 11190  9883 1307 ″ 4 9.5 15468 13993 1475  ″ 5 8.0 17118 15759 1359  ″ 6 2.0 15260 14954306 ″* 7 2.2 14000 13687 313 ″* 8 1.8 17925 17595 330 ″* *Nodenaturation

Sample 3 showed approximately 12% cross-linking, while sample 6 showedonly about 2% cross-linking, indicating an approximately 6-fold linearamplification.

9. Linear amplification using non-isotopic detection, multiple probesets, and automated cycling

Nucleic Acid Sequences

NAX 595 (SEQ ID NO: 41) 5′-TTTTTTCCAAGGAGGTAAACGCTCCTCTGB

NAX 596 (SEQ ID NO: 42 ) 5′-FATTGGTTGATCGCCCAGACAATGCAXA

NAX 601 (SEQ ID NO: 43 ) 5′-TTTTTTTCCCTTTATACGCTCAAGCAATAB

NAX 602 (SEQ ID NO: 44 ) 5′-FTCTTTGCTATAGCACTATCAAGCCAXA

NAX 607 (SEQ ID NO: 45) 5′-TTTTTTGTCTCGAACATCTGAAAGCATGGB

NAX 608 (SEQ ID NO: 46) 5′-FCTGCGTCTTGCTCTATTTGACCGCAXA

NAX 613 (SEQ ID NO: 47 ) 5′-TTTTTTTGAGCGGCTCTGTCATTTGCCCAB

NAX 614 (SEQ ID NO: 48) 5′-FTGTCCAAGGATTATTTGCTGGTCCAXA

X=ethoxycoumarin

F=fluorescein

B=biotin

Component, pmol/m Sample [mL] NAX L 1 2 3 4 595 1 0.375 0.375 596 ″0.125 0.125 601 ″ 0.375 0.375 0.375 0.375 602 ″ 0.25 0.25 0.125 0.125607 ″ 0.375 0.375 608 ″ 0.125 0.125 613 ″ 0.375 0.375 0.375 0.375 614 ″0.25 0.25 0.125 0.125 H₂O 12.75 12.75 12 12 lysis buffer* 18.5 16.5 18.516.5 target DNA** 10⁻⁵ 2 2 *Lysis buffer = 1:3 0.75M NaOH: 1X TE (pH7.5). **The target DNA is the Chlamydia cryptic plasmid cloned intopBluescript, pretreated by boiling for 30 minutes in lysis buffer.

Add 17.5 mL of neutralization buffer (1.75 mL of 3.5% BSA, 1.5 mL of 1.5M HOAc, 11.3 mL of 20×SSC, and 2.15 mL of water) to each sample, loadedin a 96-well polycarbonate plate. Add 50 mL mineral oil to preventevaporation.

The plate was put onto a programmable thermal controller beneath a bankof UV-A lamps. The thermal controller was programmed to bring thesamples through the following temperature profile: (1) 60° C. for 10minutes; (2) 85° C. for 90 seconds; (3) 58° C. for 5 minutes; (4) 55°for 5 minutes; (repeat steps 2,3,4 five times); (5) hold at 20° C. Theoperation of the bank of lamps was controlled via a control circuit thatresponds to the temperature sensed by a thermistor. The thermistor wasembedded in one of the wells in the 96-well plate. The control circuitactivated the light bank if the temperature sensed by the thermistor waswithin a narrow range (approximately ±3° C.) about a desiredtemperature, in this case 55° C.

Following the cycling procedure the mineral oil was separated from theaqueous sample, and hereafter the aqueous sample was treated to:incubation with streptavidin-coated magnetic particles, five repetitionsof removal of the supernatant liquid and addition of buffered washsolution, incubation with an anti-fluorescein/alkaline phosphataseconjugate, five repetitions of removal of the supernatant liquid andaddition of buffered wash solution, and incubation with Attophos at 37°C. The fluorescent signal generated in each sample was measured(relative fluorescence units): Sample 1, 39; 2, 142; 3, 58; 4, 250. Theresults demonstrate that a multitude of probe sets can be combined toachieve a higher signal and that the amplification process can becarried out by automated methods.

10. Nucleic acid sequence detection of Chlamyida trachomatis in clinicalsamples using an amplification probe set

Component, pmol/m Sample [mL] NAX L 1 2 3 4 601 1 1.2 1.2 1.2 1.2 602 ″0.8 0.8 0.8 0.8 lysis buffer 37 37 clinical 37 37 sample* H₂O 26 26 2626 *clinical samples were obtained by endocervical swab. The swabs wereboiled in a tube with 400 mL of lysis buffer for 30 minutes. For eachsample, 37 mL of lysate was removed for testing.

Samples 1 and 2 are from two different patients, and samples 3 and 4 arenegative controls for the experiment.

Add 35 mL of neutralization buffer (1.75 mL of 3.5% BSA, 1.5 mL of 1.5 MHOAc, 11.3 mL of 20×SSC, and 2.15 mL of water) to each sample, loaded ina 96-well polycarbonate plate. Add 50 mL mineral oil to preventevaporation.

The previous protocol was followed for amplification and detection,except that the time at 58° C. was 9 minutes and the time at 55° C. was6 minutes in each thermal cycle. The fluorescent signal generated ineach sample was measured (relative fluorescence units): Sample 1, 768;2, 43; 3, 44; 4, 53. Sample 1 was assigned as positive for the presenceof C. trachomatis and sample 2 was assigned as negative. These resultswere confirmed by PCR and culture. The results demonstrate theeffectiveness of the amplification procedure for the detection ofnucleic acid sequences in clinical specimens.

11. Geometric amplification is demonstrated in the followingexemplifications.

Nucleic Acid Sequences

NAX 441 (SEQ ID NO:36)5′-GATTTAAAAACCAAGGTCGATGTGATAGGGCTCGTATGTGGAATGTCGAACTCATCGGCGAT

NAX 443 (SEQ ID NO:37) 5′-GGGCGAGAXATATCACATCGACCTTGGTTTTTAAATC

NAX 444 (SEQ ID NO:38) 5′-GATTTAAAAACCAAGGTCGATGTGATAGGGCTCGAXAAAAA

NAX 445 (SEQ ID NO:39) 5′-TCGCCGATGAGTTCGACATTCCACATACGAGCCCTTTCTCG

NAX 446 (SEQ ID NO:40) 5′-TTTTTTTTATGTGGAATGTCGAACTCATCGGCGA

Samples, μl Component, Nax pm/μl 1 2 3 4 5 6 7 8 ³²P-443 1 1 1 1 1 1 1 11 444 1 1 1 1 1 445 .36 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 446 1 1 1 1 1441 10 fmol/μl 1 1 1 1 1 1 1 1 H₂O 9.2 9.2 9.2 9.2 9.2 9.2 9.2 9.2Conditions No. irradiations 1 3 5 7 1 3 5 7 Heat treatment Δ Δ Δ Δ Δ Δ ΔΔ

The following control samples were also run:

pm/μl 9 10 11 12 Component, Nax ³²P-443 1 1 1 1 1 444 1 1 1 445 .36 2.82.8 2.8 2.8 446 1 1 1 441 10 fmol/μl 1 1 H₂O 9.2 7.2 10.2 8.2 ConditionsNo. irradiations 7 7 7 7 Heat treatment + + Δ Δ Δ heat cycle set forthbelow + = isothermal

Protocol

Add 18.5 μl of 1:3 075 M NaOH: 1×TE to sample in a microtitre plate.

Add 17.5 μl neutralization buffer (3.5 μl of 3.5% BSA; 1.5 μl of 1.5MHOAc; 11.3 μl of 20×SSC and 0.4 μl of water) to each well.

Layer 50 μl mineral oil on top of each well to inhibit evaporation.

Incubate 20 minutes at 40° C.

Irradiate at 40° C. for 20 minutes. (UV-A light source)

Denature for 2 minutes at 90° C.

Analysis by 10% PAGE with 7M urea.

Bands were excised and the amount of ³²P in each band was quantified byscintillation counting.

Results

The results are summarized in the following table.

Counts in Counts in % Conversion to Sample Total Counts StartingMaterial Product Product 1 5218 5201 17 0.3 2 5437 5382 55 1.0 3 50835019 64 1.3 4 5156 5081 75 1.6 5 4846 4827 19 0.4 6 4777 4708 69 1.4 74859 4706 153 3.1 8 4830 4471 359 7.4 9 5629 5616 13 0.2 10 5486 5429 571.0 11 5548 5543 5 1 12 5536 5517 19 0.3

The results demonstrate that by employing two complementary sets ofprobes, a geometric amplification of the signal indicative of thepresence of the target nucleic acid may be obtained.

12. The Use of a Fith Probe as a Protective System is DemonstratedNucleic Acid Sequences:

NAX 442 (SEQ ID NO:49)5′-ATCGCCGATGAGTTCGACATTCCACATACGAGCCCTATCACATCGACCTT GGTTTTTAAATC

NAX562 (SEQ ID NO:50) 5′-AAAGGGCTCGAAAAA

Component, Sample [μl] NAX pmol/μl 1 2 3 4 ³²P-446 0.52† 1.9 1.9 1.9 1.9443 1 1 1 1 1 444 1 1 1 1 1 445 1 1 1 1 1 562 1 1 562 10 1 1 442 10⁻⁴ 1H₂O 9.1 8.1 8.1 7.1 †prepared from 1.0 μl of 0.1 pmol/μl ³²P-446 and 0.9μl of 1.0 pmol/μl 446

Protocol:

Add 18.5 μl of 1:3 0.75M NaOH: 1×TE (pH7.5) to each sample, loaded in a96-well microtitre plate.

Add 17.5 μl of neurtalization buffer (1.75 μl of 3.5% BSA, 1.5 μl of 1.5M HOAc, 11.3 μl of 20×SSC, and 2.15 μl of water) to each sample. Add 50μl mineral oil to prevent evaporation.

Incubate at 55° C. for 15 minutes.

Perform 30 cycles of: incubated at 46° C. for 1 minute; irradiate withUV-A light at 43° C. for 7 minutes: and denature at 90° C. for 1 minute.

The samples analyzed by denaturing PAGE (13% with 7M urea).

The degree of product formed (NAX 446 crosslinked to NAX 444) asobserved by gel electrophoresis and quantification by scintillationcounting was: 1, 8.0%; 2, 2.7%; 3,1.3%; 4,23%. The results demonstratethat the fifth probe (NAX 562 suppresses the occurrence of a targetindependent reaction, and does not prevent the target specificamplification from occurring.

13. Chemical Amplification Using a Coordination Complex as theCrosslinker

Another class of crosslinking agents that are useful for covalentlycrosslinking two probes comprises metal coordination complexes.Activation of the metal complex may be either photonic or thermal. Theactivated complex may then react by substitution, addition, orcyclization with an appropriate reactant situated on the opposite strandin the stem, and the two probes are covalently crosslinked as a resultof the new coordination complex produced.

For example, platinum(II) complexes are useful for forming complexeswith amine ligands as well as nucleic acid bases, especially guanine andadenine. These complexes undergo thermal substitution reactions, andsquare planar Pt(II) complexes are known to photodissociate upon UVirradiation and subsequently add a ligand.

Photocrosslinking

A crosslinker probe is prepared with a platinum complex adduct at aspecific site in the stem region, and a recipient probe is prepared withan appropriate ligand to react with the photoactivated complex, forexample, an alkylamine, spatially situated for optimal contact with theplatinum complex.

Example. Probes with the following sequences are prepared:

NAXP 019 (SEQ ID NO: 51)5′-TCTTTATTTAGATATAGAATTTCTTTTTTAGAGAGTTTAGAAGAAT

NAXP 020 (SEQ ID. 52) 5′-ATTCTTCTAAACTCTCTAAAAAACAAG′G′AA

NAXP 021 (SEQ ID. 53 ) 5′-TT*CCT*TGGAAATTCTATATCTAAATAAAGA

NAXP 022 (SEQ ID. 54) 5′-ATTCTTCTAAACTCTCTAAAAAACAAG′AA

NAXP 023 (SEQ ID.55) 5′-TT*CT*TGGAAATTCTATATCTAAATAAAGA

T*=amine ligand-containing base: 2′-deoxy-5-(b-aminoethoxymethyl)uridine

G′=site of Pt adduct

Underlined bases comprise the stem-forming portion of theoligonucleotide

NAXP 019 is homologous to the − strand of the Chlamydia cryptic plasmid,complementary to the + strand, postion 3878-3900.

Preparation of recipient probes (NAXP 021, NAXP 023). The amineligand-containing base is prepared according to Baker et.al., J. Med.Chem. (1966), 9, 66, from 2′-deoxyuridine andN-trifluoroacetyl-2-aminoethanol. The fully protectedN-trifluoroacetyl-5′-O-dimethoxytrityl-3′-O-phosphoramidite is preparedby standard techniques. The oligonucleotide is then prepared by standardautomated synthesis techniques. Deprotection of the aminoethyl etheroccurs during deprotection of the oligonucleotide by treatment with 40%aqueous ammonia. The oligonucleotide is isolated by denaturingpolyacrylamide gel electrophoresis. The band containing the product isexcised, extracted into water, and purified and desalted by passagethrough a Sephadex G25 column. The oligonucleotide is reconstituted in aknown volume of distilled water and the concentration determined by theabsorbance at 260 nm.

Preparation of crosslinker probes (NAXP 020, NAXP 022). In NAXP 020,G′G′ represents the bidentate adductcis-[Pt(NH₃)₂{d(GpG)-N7(G₂₇),-N7(G₂₈)}], and in NAXP 022, G′ representsthe monodentate adduct [Pt(NH₃)₃{d(G)-N7(G₂₇)}]. NAXP020 is prepared bythe reaction of the oligonucleotide (purified as stated above) with thediaqua compound cis-[Pt(NH₃)₂(H₂O)₂]²⁺ at 37° C. for 18 hours, and NAXP022 is prepared by the reaction of the oligonucleotide (purified asstated above) with the monoaqua compound [Pt(NH₃)₃(H₂O)]²⁺ at 37° C. for18 hours. Each of the products is obtained by anion exchange HPLC anddesalted by dialysis.

The ability to form crosslinks with a Pt adduct between probes in atemplated reaction

Component, pmol/m Sample [mL] NAXP L 1 2 3 4 5 6 7 8 9 ³²P-019 0.2 1 1 1³²P-020 0.2 1 1 1 ³²P-021 0.2 1 1 1 019 1 1 1 1 1 020 1 1 1 1 1 021 1 11 1 1 H₂O 12  12  12  12  12  12  11  11  11 

Protocol

Add 18.5 mL of 1:3 0.75M NaOH: 1×TE (pH 7.5) to each sample, loaded in a96-well microtitre plate.

Add 17.5 mL of neutralization buffer (1.75 mL of 3.5% BSA, 1.5 mL of 1.5M HOAc, 11.3 mL of 20×SSC, and 2.15 mL of water) to each sample. Add 60mL mineral oil to prevent evaporation.

Incubate at 40° C. for 15 minutes.

Irradiate at 40° C. for 20 minutes using UV-A lamps (sharp cut-offfilter at 300 nm)

Analyze by denaturing PAGE (15% with 7 M urea)

The effect of thermal cycling on the amount of crosslinked productformed

Component, pmol/m Sample [mL] NAXP L 1 2 3 4 5 6 7 8 ³²P-021 0.2  1  1 1  1  1  1  1  1 019 0.01  1  1  1  1 020 1  1  1  1  1  1  1  1  1 0211  1  1  1  1  1  1  1  1 H₂O 11 11 10 10 11 11 10 10 No. cycles  1  2 1  2  5  5  5  5 thermal + D + D + D + D treat. + = isothermal, nodenaturation D = samples denatured each cycle

Protocol:

The sample preparation is the same as above. After an initial incubationfor 10 minutes at 40° C. the samples were treated as indicated in thetable.

Cycle:

Irradiate at 40° C. for 10 minutes

Heat to 85° C. for 2 minutes

Incubate at 40° C. for 10 minutes

Repeat the cycle procedure the indicated number of times, ending withthe irradiation step at that cycle number.

Analyze by denaturing PAGE (15%, with 7 M urea)

The analogous set of experiments are performed using the monodentateadduct as the crosslinking probe, NAXP 022, the recipient probe NAXP023, and the synthetic target NAXP 019.

Thermal crosslinking reaction

A crosslinker probe is prepared with a platinum complex adduct at aspecific site in the stem region, and a recipient probe is prepared withan appropriate ligand to react with the complex, for example, asulfur-containing ligand, spatially situated for optimal contact withthe platinum complex.

Example. Probes with the following sequences are prepared:

NAXP 024 (SEQ ID. 56 ) 5′-ATTCTTCTAAACTCTCTAAAAAA

NAXP 025 (SEQ ID. 57)

5′-TTLTTGGAAATTCTATATCTAAATAAAGA

M=a Pt or Pd square planar complex, (L₃)MX, where L₃ is a tridentateligand with linking arm joined to the oligo backbone and X is a ligandchosen from OH₂, Cl⁻, Br⁻, I⁻, N₃ ⁻, SCN⁻, NO₂ ⁻, NH₃, pyridine, and thelike. L₃ may be a terpyridinyl or diethylenetriamine derivative.

L=4-thiouridine, 2′-deoxy-4-thiouridine, 4-thiothymidine, (the2,4-dithio analogues of these), non-nucleosidic group containing amercapto group.

Include excess X in the solution to suppress substition reactions at themetal complex when it is not hybridized in the stem. The rate of thesubstitution reaction can be varied by the choice of the metal, (Pdfaster than Pt), or the choice of the fourth ligand X (reactivityfollows in the order listed above, fastest to slowest).

Except for the irradiation step, the procedure will be substantially thesame as for the photoactivation.

13. Chemical Amplification Using an Organometallic Complex as theCrosslinker

Another class of crosslinking agents that are useful for covalentlycrosslinking two probes comprises organometallic complexes. Activationof the metal complex may be photonic, and the activated complex may thenreact by substitution with an appropriate reactant situated on theopposite strand in the stem, and the two probes are covalentlycrosslinked as a result of the new bond formed.

For example, cyclopentadienyl manganese(I) complexes, CpMnL₃, where L isa neutral two electron donor ligand such as CO, are useful for theirrich photochemical reactivity. These complexes, in contrast, are inertto thermal substitution reaction conditions and thus provide a systemthat selectively responds to photonic activation. Photoirradiation using300-350 nm light induces the loss of a CO ligand. The intermediate,CpMnL₂, can recombine with the extruded ligand or react with anothersuitable ligand, L′, such as a phosphine, phosphite, amine, ether,olefin, etc. The photoreactivity of the newly formed compound depends onthe identity of the new ligand. When L′ is a phosphine or phosphite anysubsequent reactions proceed with loss of another CO ligand; thephosphine or phosphite remains bound to the metal. In contrast, for mostother ligands L′ it is this ligand that is photosubstituted upon furtherreaction.

Example. Probes with the following sequences are prepared:

NAXM 011 (SEQ ID NO: 58) 5′-GATACGACGCCGCAAAAGCTCTTCATMAG

NAXM 012 (SEQ ID NO:59 ) 5′-CTLATCCAAGCCGAGTCTACAGTTATAGG NAXM 013 (SEQID NO:60) 5′-CCTATAACTGTAGACTCGGCTTGGGAAGAGCTTTTGCGGCGTCGTATC

M=cyclopentadienylmanganese(I) tricarbonyl

L=trialkylphosphite

Underlined bases comprise the stem-forming portion of theoligonucleotide

Preparation of NAXM 011. Lithium cyclopentadienide is condensed with2,2-dimethyl-1,3-dioxolane-4-methyl mesylate. The trimethyltin adduct ofthe cyclopentadiene derivative is reacted with Mn(CO)₅Br to yield2,2-dimethyl-1,3-dioxolane-4-methylcyclopentadienylmanganesetricarbonyl. The ketal is hydrolyzed to produce1-(cyclopentadienylmanganese tricarbonyl)-2,3-propanediol. The diol isconverted to the 3-O-dimethoxytrityl-2-O-phosphoramidite derivative andthe title modified oligonucleotide is prepared by automated DNAsynthesis techniques.

Preparation of NAXM 012

The di-t-butylsilylene of 1,1,1-tris(hydroxymethyl)ethane is preparedand the third hydroxyl group is protected as the p-nitrobenzyl ether.The silylene is selectively hydroyzed using tributylammonium fluoride toproduce 2-methyl-2-(methyl p-nitrobenzyl ether)-1,3-propanediol. Thediol is converted to the 1-O-dimethoxytrityl-3-O-phosphoramiditederivative and the title sequence is prepared by automated DNA synthesistechniques. The oligo is cleaved from the solid support without removingthe protecting groups from the exocyclic amines or the phosphate groups.The solution is irradiated with 320 nm light to remove the p-nitrobenzylether protecting group. The oligo is lyophilized, dissolved in anhydrousacetonitrile and reacted with diethyl chlorophosphite. Theoligonucleotide is then fully deprotected by treatment with 40% aqueousammonia, isolated by reverse phase HPLC and purified by passage througha Sephadex G25 column.

The ability to form crosslinks via a templated photosubstitutionreaction.

Component, pmol/m Sample [mL] NAXM L 1 2 3 4 5 6 7 8 9 ³²P-011 0.2 1 1 1³²P-012 0.2 1 1 1 ³²P-013 0.2 1 1 1 011 1 1 1 1 1 012 1 1 1 1 1 013 1 11 1 1 H₂O 11  11  11  11  11  11  10  10  10 

Protocol:

Add 1 mL of 50 mM 2-aminoethanol to each sample, loaded in a 96-wellmicrotitre plate.

Add 18.5 mL of 1:3 0.75M NaOH: 1×TE (pH 7.5) to each sample

Add 17.5 mL of neutralization buffer (1.75 mL of 3.5% BSA, 1.5 mL of 1.5M HOAc, 11.3 mL of 20×SSC, and 2.15 mL of water) to each sample. Add

60 mL mineral oil to prevent evaporation.

Incubate at 40° C. for 15 minutes.

Irradiate at 40° C. for 20 minutes using UV-A lamps (sharp cut-offfilter at 300 nm)

Analyze by denaturing PAGE (15% with 7 M urea)

The effect of thermal cycling on the amount of crosslinked productformed

Component, pmol/m Sample [mL] NAXM L 1 2 3 4 5 6 7 8 ³²P-012 0.2 1  1 1 1 1 1 1  1 013 0.01 1  1 1  1 011 1 1  1 1  1 1 1 1  1 012 1 1  1 1  11 1 1  1 H₂O 10  10 9 10 10  9 9 10 No. cycles 1  2 1  2 5 5 5  5thermal + D + D + D + D treat. + = isothermal, no denaturation D =samples denatured each cycle

Protocol:

The sample preparation is the same as above. After an initial incubationfor 10 minutes at 40° C. the samples were treated as indicated in thetable.

Cycle:

Irradiate at 40° C. for 10 minutes

Heat to 85° C. for 2 minutes

Incubate at 40° C. for 10 minutes

Repeat the cycle procedure the indicated number of times, ending withthe irradiation step at that cycle number.

Analyze by denaturing PAGE (15%, with 7 M urea)

It is evident from the above results that the subject methodologyprovides a convenient and efficient way to identify the presence ofspecific nucleic acid sequences. Amplification is achieved in theabsence of enzyme, using chemical reactions to cross-link two probestethered together by means of a template. Once the two probes have beencross-linked, they in turn may serve as a template for homologoussequences. In this way, a geometric expansion of cross-linked probes maybe obtained in relation to a target sequence. Use of the subjectautomatic devices for performing the subject assays provides forminimization of error introduction and improved consistency in assayconditions.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

60 25 base pairs nucleic acid single linear other nucleic acid /desc =“probe” unknown 1 GTTTTTCTTG TTGAACAAAA ATCCT 25 26 base pairs nucleicacid single linear other nucleic acid /desc = “probe” unknown 2TTTCTAGGGG GAACACCCGT GTGTCT 26 60 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown 3 CTGGGAAACA TCAAAGGAATTCTCGGAAAG AAAGCCAGCA GTCTCCTCTT TACAGAAAAG 60 26 base pairs nucleicacid single linear other nucleic acid /desc = “probe” unknown 4CATAGGGGAT ACCAGACAAT TCAAAC 26 25 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown 5 ACCTGACCGT GCGCCCTTCA CAAAG25 25 base pairs nucleic acid single linear other nucleic acid /desc =“probe” unknown 6 ACGATGCCGC CATCCTCCTG CAAAA 25 26 base pairs nucleicacid single linear other nucleic acid /desc = “probe” unknown 7CACAGACCAT TCGCAGTATT GAAAAC 26 30 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown 8 ATTTTGTCTT TGCGCACAGACGATCTATTT 30 22 base pairs nucleic acid single linear other nucleicacid /desc = “probe” unknown 9 AAATAGATCG TCTGTTTGCT TT 22 20 base pairsnucleic acid single linear other nucleic acid /desc = “probe” unknownmisc_feature 1..20 /label= N /note= “N=ethoxycoumarin” 10 ANAGCGCGCAAAGACAAAAT 20 21 base pairs nucleic acid single linear other nucleicacid /desc = “probe” unknown 11 ATTTTGTCTT TGCGCGGCTT T 21 22 base pairsnucleic acid single linear other nucleic acid /desc = “probe” unknownmisc_feature 21 /note= “N=ethoxycoumarin” 12 AAATAGATCG TCTGTTTGCA NA 2220 base pairs nucleic acid single linear other nucleic acid /desc =“probe” unknown 13 TTTGCGCGCA AAGACAAAAT 20 48 base pairs nucleic acidsingle linear other nucleic acid /desc = “probe” unknown 14 TTTATAAAAAGCTCGTAATA TGCAAGAGCA TTGTAAGCAG AAGACTTA 48 32 base pairs nucleic acidsingle linear other nucleic acid /desc = “probe” unknown 15 TTTATAAAAAGCTCGTAATA TGCTTTTTTT TT 32 32 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown 16 TAAGTCTTCT GCTTACAATGCTCTTTTTTT TT 32 26 base pairs nucleic acid single linear other nucleicacid /desc = “probe” unknown misc_feature /note= “N=ethoxycoumarin” 17ANAGCATATT ACGAGCTTTT TATAAA 26 28 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown misc_feature /note= “N =ethoxycoumarin” 18 ANAAAGCATA TTACGAGCTT TTTATAAA 28 30 base pairsnucleic acid single linear other nucleic acid /desc = “probe” unknownmisc_feature /note= “N=ethoxycoumarin” 19 AAAANAAGCA TATTACGAGCTTTTTATAAA 30 30 base pairs nucleic acid single linear other nucleicacid /desc = “probe” unknown misc_feature /note= “N=ethoxycoumarin” 20ANAAAAAGCA TATTACGAGC TTTTTATAAA 30 48 base pairs nucleic acid singlelinear other nucleic acid /desc = “probe” unknown 21 TAAGTCTTCTGCTTACAATG CTCTTGCATA TTACGAGCTT TTTATAAA 48 49 base pairs nucleic acidsingle linear other nucleic acid /desc = “probe” unknown 22 TAAGTCTTCTGCTTACAATG AACTTGCATA TTACGAGCTT TTTATAAAT 49 35 base pairs nucleic acidsingle linear other nucleic acid /desc = “probe” unknown 23 TTTTTTTTTCTTGCATATTA CGAGCTTTTT ATAAA 35 29 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown 24 TTTTTTTTTC ATTGTAAGCAGAAGACTTA 29 33 base pairs nucleic acid single linear other nucleic acid/desc = “probe” unknown misc_feature 29 /note= “N=ethoxycoumarin” 25TTTATAAAAA GCTCGTAATA TGCAAGAANA AAA 33 33 base pairs nucleic acidsingle linear other nucleic acid /desc = “probe” unknown misc_feature 28/note= “N=ethoxycoumarin” 26 TTTATAAAAA GCTCGTAATA TGCAAGANAA AAA 33 54base pairs nucleic acid single linear other nucleic acid /desc = “probe”unknown 27 GATATCGGAT TTACCAAATA CGGCGGGCCC GCCGTTAGCT AACGCTAATC GATT54 31 base pairs nucleic acid single linear other nucleic acid /desc =“probe” unknown misc_feature /note= “N=ethoxycoumarin” 28 AAAAANAGCCGTTAGCTAAC GCTAATCGAT T 31 37 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown 29 GATATCGGAT TTACCAAATACGGCGGGCCC TTTTTTT 37 31 base pairs nucleic acid single linear othernucleic acid /desc = “probe” unknown misc_feature /note=“N=ethoxycoumarin” 30 AAAAANAGCC GTATTTGGTA AATCCGATAT C 31 37 basepairs nucleic acid single linear other nucleic acid /desc = “probe”unknown 31 AATCGATTAG CGTTAGCTAA CGGCGGGCCC TTTTTTT 37 30 base pairsnucleic acid single linear other nucleic acid /desc = “probe” unknownmisc_feature /note= “N=ethoxycoumarin” 32 AAANAAGCCG TTAGCTAACGCTAATCGATT 30 30 base pairs nucleic acid single linear other nucleicacid /desc = “probe” unknown misc_feature /note= “N=ethoxycoumarin” 33AANAAAGCCG TTAGCTAACG CTAATCGATT 30 30 base pairs nucleic acid singlelinear other nucleic acid /desc = “probe” unknown misc_feature /note=“N=ethoxycoumarin” 34 ANAAAAGCCG TTAGCTAACG CTAATCGATT 30 30 base pairsnucleic acid single linear other nucleic acid /desc = “probe” unknownmisc_feature /note= “N=ethoxycoumarin” 35 NAAAAAGCCG TTAGCTAACGCTAATCGATT 30 62 base pairs nucleic acid single linear other nucleicacid /desc = “probe” unknown 36 GATTTAAAAA CCAAGGTCGA TGTGATAGGGCTCGTATGTG GAATGTCGAA CTCATCGGCG 60 AT 62 37 base pairs nucleic acidsingle linear other nucleic acid /desc = “probe” unknown misc_feature/note= “N=ethoxycoumarin” 37 GGGCGAGANA TATCACATCG ACCTTGGTTT TTAAATC 3741 base pairs nucleic acid single linear other nucleic acid /desc =“probe” unknown misc_feature 36 /note= “N=ethoxycoumarin” 38 TTAAAAACCAAGGTCGA TGTGATAGGG CTCGANAAAA A 41 41 base pairs nucleic acid singlelinear other nucleic acid /desc = “probe” unknown 39 TCGCCGATGAGTTCGACATT CCACATACGA GCCCTTTCTC G 41 34 base pairs nucleic acid singlelinear other nucleic acid /desc = “probe” unknown 40 TTTTTTTTATGTGGAATGTC GAACTCATCG GCGA 34 30 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown misc_feature 30 /note=“N=biotin” 41 TTTTTTCCAA GGAGGTAAAC GCTCCTCTGN 30 28 base pairs nucleicacid single linear other nucleic acid /desc = “probe” unknownmisc_feature /note= “N=fluorescein” misc_feature 27 /note=“N=ethoxycoumarin” 42 NATTGGTTGA TCGCCCAGAC AATGCANA 28 30 base pairsnucleic acid single linear other nucleic acid /desc = “probe” unknownmisc_feature 30 /note= “N=biotin” 43 TTTTTTTCCC TTTATACGCT CAAGCAATAN 3028 base pairs nucleic acid single linear other nucleic acid /desc =“probe” unknown misc_feature /note= “n=fluorescein” misc_feature 27/note= “N=ethoxycoumarin” 44 NTCTTTGCTA TAGCACTATC AAGCCANA 28 30 basepairs nucleic acid single linear other nucleic acid /desc = “probe”unknown misc_feature 30 /note= “N=biotin” 45 TTTTTTGTCT CGAACATCTGAAAGCATGGN 30 28 base pairs nucleic acid single linear other nucleicacid /desc = “probe” unknown misc_feature /note= “N=fluorescein”misc_feature 27 /note= “N=ethoxycoumarin” 46 NCTGCGTCTT GCTCTATTTGACCGCANA 28 30 base pairs nucleic acid single linear other nucleic acid/desc = “probe” unknown misc_feature 30 /note= “n=biotin” 47 TTTTTTTGAGCGGCTCTGTC ATTTGCCCAN 30 28 base pairs nucleic acid single linear othernucleic acid /desc = “probe” unknown misc_feature /note= “N=fluorescein”misc_feature 27 /note= “N=ethoxycoumarin” 48 NTGTCCAAGG ATTATTTGCTGGTCCANA 28 62 base pairs nucleic acid single linear other nucleic acid/desc = “probe” unknown 49 ATCGCCGATG AGTTCGACAT TCCACATACG AGCCCTATCACATCGACCTT GGTTTTTAAA 60 TC 62 15 base pairs nucleic acid single linearother nucleic acid /desc = “probe” unknown 50 AAAGGGCTCG AAAAA 15 46base pairs nucleic acid single linear other nucleic acid /desc = “probe”unknown 51 TCTTTATTTA GATATAGAAT TTCTTTTTTA GAGAGTTTAG AAGAAT 46 30 basepairs nucleic acid single linear other nucleic acid /desc = “probe”unknown 52 ATTCTTCTAA ACTCTCTAAA AAACAAGGAA 30 30 base pairs nucleicacid single linear other nucleic acid /desc = “probe” unknownmisc_feature /note= “N=2′-deoxy-5-(b-aminoethoxymethyl)uridine”misc_feature /note= “N=2′-deoxy-5(b-aminoethoxymethyl)uridine” 53TNCCNTGGAA ATTCTATATC TAAATAAAGA 30 29 base pairs nucleic acid singlelinear other nucleic acid /desc = “probe” unknown 54 ATTCTTCTAAACTCTCTAAA AAACAAGAA 29 29 base pairs nucleic acid single linear othernucleic acid /desc = “probe” unknown misc_feature /note=“N=2′-deoxy-5-(b-aminoethoxymethyl)uridine” misc_feature /note=“N=2′-deoxy-5-(b-aminoethoxymethyl)uridine” 55 TNCNTGGAAA TTCTATATCTAAATAAAGA 29 29 base pairs nucleic acid single linear other nucleic acid/desc = “probe” unknown misc_feature 27 /note= “N=Pt or Pd square planarcomplex” 56 ATTCTTCTAA ACTCTCTAAA AAACAANAA 29 29 base pairs nucleicacid single linear other nucleic acid /desc = “probe” unknownmisc_feature /note= “N=4-thiouridine” 57 TTNTTGGAAA TTCTATATCT AAATAAAGA29 29 base pairs nucleic acid single linear other nucleic acid /desc =“probe” unknown misc_feature 27 /note=“N=cyclopentadienylmanganese(I)tricarbonyl” 58 GATACGACGC CGCAAAAGCTCTTCATNAG 29 29 base pairs nucleic acid single linear other nucleic acid/desc = “probe” unknown misc_feature /note= “N=trialkylphosphate” 59CTNATCCAAG CCGAGTCTAC AGTTATAGG 29 48 base pairs nucleic acid singlelinear other nucleic acid /desc = “probe” unknown 60 CCTATAACTGTAGACTCGGC TTGGGAAGAG CTTTTGCGGC GTCGTATC 48

What is claimed is:
 1. A method for detecting a target nucleic acidsequence in a sample, said method employing at least one pair of probescharacterized by having sequences homologous to adjacent portions ofsaid target nucleic acid sequence and each probe having at least oneside chain which non-covalently binds to a side chain of the other probeof the pair to form a stem upon base pairing of said probes to saidtarget nucleic acid sequence, at least one of said side chains having anactivatable group, which upon activation during stem formation forms acovalent cross-link with the other side chain member of said stem, saidmethod comprising: a) combining said sample with said at least one pairof said probes under conditions of base pairing between said probes andsaid target nucleic acid to produce an assay medium, whereby probesbinding to said target nucleic acid form said stem; b) activating saidactivatable group, whereby a covalent cross-link occurs between saidside chain members of said stem; and c) detecting the presence ofcross-linked pairs of probes as indicative of the presence of saidtarget sequence in said sample.
 2. A method according to claim 1,wherein said activating is photoactivating.
 3. A method according toclaim 1, wherein said target nucleic acid is double stranded and twodifferent pairs of probes are used, where each pair is homologous to oneof the strands of said target nucleic acid.
 4. A method for detecting atarget nucleic acid sequence in a sample, said method employing at leastone pair of probes characterized by having sequences homologous toadjacent portions of said target nucleic acid sequence and each probehaving at least one side chain which non-covalently binds to a sidechain of the other probe of the pair to form a stem upon base pairing ofsaid probes to said target nucleic acid sequence, at least one of saidside chains having a photoactivatable group, which upon activationduring stem formation forms a covalent cross-link with the other sidechain member of said stem, each of said side chains having at least twonucleotides capable of base pairing with the other side chain to formsaid stem, said method comprising: a) combining said sample with said atleast one pair of said probes under conditions of base pairing betweensaid probes and said target nucleic acid, wherein when said targetnucleic acid is single stranded, at least a first pair of probes isadded which is homologous to said single stranded nucleic acid, and ifsaid target nucleic acid is double stranded, at least one of first andsecond pairs of probes are added, which pairs are homologous to one orthe other strands of said double stranded target nucleic acid, wherebyprobes binding to said target nucleic acid form said stem; b)photoactivating said photoactivatable group, whereby a covalentcross-link is formed between said side chain members of said stem; c)melting double stranded nucleic acid; d) repeating the following cycleat least once: 1) incubating for sufficient time for base pairingbetween homologous sequences to occur, with the proviso that when onlysaid first pair of probes was added, another pair of probes is addedhaving sequences homologous to said first pair of probes; 2)photoactivating said photoactivatable group, whereby a covalentcross-link is formed between said side chain members of said stem; and3) melting double stranded DNA, which ends a cycle; and e) detecting thepresence of cross-linked pairs of probes as indicative of the presenceof said target sequence in said sample.
 5. A method according to claim4, wherein said target sequence has a gap of fewer than 2 nucleotidesbetween the sequences homologous to said pair of probes and each of saidside chains of said stem comprise at least three nucleotides orhybridizable analogs thereof which form base pairs.
 6. A methodaccording to claim 5, wherein said side chains have from 3 to 8nucleotides or hybridizable analogs thereof which base pair to form saidstem.
 7. A method according to claim 4, wherein said photoactivatablegroup reacts with a nucleotide or analog thereof to form a covalent bondcross-link.
 8. A method according to claim 7, wherein saidphotoactivatable group is a moiety comprising a coumarin orfurocoumarin.
 9. A method according to claim 4, wherein on one of saidside chains said at least two nucleotides capable of base pairing withthe other side chain to form said stem are separated from saidphotoactivatable group.
 10. A method according to claim 4, wherein eachof said side chains comprises different fluorophores, wherein the energyof emitted light of a first of said fluorophores is in the absorptionband of a second of said fluorophores, wherein said detecting thepresence of cross-linked probes comprises: exciting said firstfluorophore; and reading the fluorescence of said second fluorophore.11. A method according to claim 4, wherein each of probes comprises alabel, wherein one of said labels is a member of a specific binding pairand the other of said labels provides for a detectable signal, whereinsaid detecting the presence of cross-linked probes comprises: separatingsaid cross-linked probes from said sample on a solid support; anddetecting the presence of said signal on said solid support.
 12. Amethod according to claim 4, wherein at least three cycles are repeated.13. A method for detecting a sequence of target dsDNA in a sample, saidmethod employing first and second pairs of probes characterized byhaving sequences homologous to adjacent portions of first and secondstrands of said target dsDNA and each probe having at least one sidechain which non-covalently binds to a side chain of the other probe ofthe pair to form a stem upon base pairing of said probes to said targetfirst and second strands, respectively, at least one of said side chainsin each pair having a photoactivatable group, which upon activationduring stem formation forms a covalent cross-link with the other sidechain member of said stem, each of said side chains having at least twonucleotides capable of base pairing with the other side chain to formsaid stem, said method comprising: a) combining said sample with saidfirst and second pairs of probes under conditions of base pairingbetween said probes and said target nucleic acid, which first and secondpairs of probes are homologous to one or the other strands of saidtarget dsDNA, whereby probes binding to said target nucleic acid formsaid stem; b) photoactivating said photoactivatable group, whereby acovalent cross-link isformed between said side chain members of saidstem; c) melting double stranded nucleic acid; d) repeating thefollowing cycle at least once: 1) incubating for sufficient time forbase pairing between homologous sequences to occur; 2) photoactivatingsaid photoactivatable group, whereby a covalent cross-link occursbetween said side chain members of said stem; and 3) melting doublestranded DNA, which ends a cycle; and e) detecting the presence ofcross-linked pairs of probes as indicative of the presence of saidtarget dsDNA in said sample.
 14. A method according to claim 13, whereinone of said side chains has a bulge between the last nucleotide of saidside chain base pairing with said target sequence and the firstnucleotide of said side chain base pairing with a nucleotide of theother side chain member of said stem.
 15. A method according to claim13, wherein said side chains have from 3 to 8 nucleotides which basepair to form said stem.
 16. A method according to claim 13, wherein saidphotoactivatable group reacts with a nucleotide or analog thereof toform a covalent bond cross-link.
 17. A method according to claim 13,wherein said photoactivatable group is a moiety comprising a coumarin orfurocoumarin.
 18. A method according to claim 13, wherein at least threecycles are repeated.
 19. A method according to claim 13, wherein each ofsaid side chains comprises different fluorophores, wherein the energy ofemitted light of a first of said fluorophores is in the absorption bandof a second of said fluorophores, wherein said detecting the presence ofcross-linked probes comprises: exciting said first fluorophore; andreading the fluorescence of said second fluorophore.
 20. A methodaccording to claim 13, wherein each of said probes comprises a label,wherein one of said labels is a member of a specific binding pair andthe other of said labels provides for a detectable signal, wherein saiddetecting the presence of cross-linked probes comprises: separating saidcross-linked probes from said sample on a solid support; and detectingthe presence of said signal on said solid support.
 21. A method fordetecting a target nucleic acid sequence in a sample, said methodemploying at least one pair of probes characterized by having sequenceshomologous to adjacent portions of said target nucleic acid sequence andeach probe having at least one side chain which non-covalently binds toa side chain of the other probe of the pair to form a stem upon basepairing of said probes to said target nucleic acid sequence, at leastone of said side chains having an activatable group, which uponactivation during stem formation forms a covalent cross-link with theother side chain member of said stem, and at least one of said stemsforming a hairpin or stem and loop by one portion of said side chainbinding to a different portion of said chain or to the sequence of saidprobe homologous to said target, said method comprising: a) combiningsaid sample with said pair of probes under conditions of melting of saidhairpin or stem and loop and of base pairing between said probes andsaid target nucleic acid to produce an assay medium, whereby probesbinding to said target nucleic acid form said stem; b) activating saidactivatable group, whereby a covalent cross-link occurs between saidside chain members of said stem; and c) detecting the presence ofcross-linked pairs of probes as indicative of the presence of saidtarget sequence in said sample.
 22. A method according to claim 21,wherein said hairpin comprises a bulge, said bulge comprising saidphotoactivatable group.
 23. A method according to claim 21, wherein oneof said side chains comprises a terminal sequence complementary to thesequence homologous to said target sequence joined to said stem formingsequence by a linking group other than an oligonucleotide.
 24. A kitcomprising at least one pair of probes, said probes being characterizedby having sequences homologous to adjacent portions of a target nucleicacid sequence and each probe having at least one side chain whichnon-covalently binds to a side chain of the other probe of the pair toform a stem upon base pairing of said probes to said target nucleic acidsequence, at least one of said side chains having an activatable group,which upon activation during stem formation forms a covalent cross-linkwith the other side chain member of said stem.
 25. A kit according toclaim 24, comprising at least two pairs of probes, wherein said targetnucleic acid sequence is single or double stranded nucleic acid and thesequences of said probes homologous to one strand of said nucleic acidof one pair of probes are homologous to the sequences of the other pairof probes.
 26. A kit according to claim 25, wherein each of the membersof said stem have at least two nucleotides which base pair to form saidstem and one member of each of said pairs of probes has a bulge in theside chain between the last nucleotide of said side chain base pairingwith said target sequence and the first nucleotide of said side chainbase pairing with a nucleotide of the other side chain member of saidstem.
 27. A kit according to claim 24, wherein at least one of said sidechains which comprises an activatable group forms a hairpin or stem andloop by one portion of said side chain binding to a different portion ofsaid side chain or to the sequence of said probe homologous to saidtarget.
 28. A nucleic acid compound comprising a nucleic acid sequenceof at least 12 nucleotides defining a sequence of interest covalentlylinked at one end to a side chain characterized by having at least twonucleotides and not more than 8 nucleotides and having aphotoactivatable group other than a nucleotide.
 29. A nucleic acidcompound according to claim 28, wherein said photoactivatable group is amoiety comprising a coumarin or furocoumarin covalently bonded to alinking group in the backbone of said nucleic acid compound andcomprising other than a nucleoside.
 30. An oligonucleotide comprisingfrom 2 to 60 nucleotides and a deoxyribosyl backbone having interveningin said backbone from 1 to 2 linkers comprising other than nucleosidesand pendent from said linkers, a photoactivatable group other than anucleotide, capable of forming a covalent bond with a nucleotide.
 31. Anoligonucleotide according to claim 30, wherein said oligonucleotide is amember of a specific binding pair.
 32. An oligonucleotide according toclaim 30, wherein said oligonucleotide further comprises a directlydetectable label.
 33. An oligonucleotide according to claim 32, whereinsaid directly detectable label is a fluorophore.
 34. An oligonucleotideaccording to claim 30, wherein said photoactivatable group comprisescoumarin.